CN118059111A - Application of MDL-800 in myocardial ischemia reperfusion injury of mice - Google Patents
Application of MDL-800 in myocardial ischemia reperfusion injury of mice Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedicine, and discloses application of MDL-800 in myocardial ischemia reperfusion injury of mice, wherein the MDL-800 adopted by the invention is a class I Hdac6 small molecule agonist newly developed in the market, and is purchased from the market, and the animals adopt C57BL/6J mice; myocardial ischemia reperfusion animal model; detecting the heart contraction function of the mice by using the ultrasound of the small animals; detecting myocardial infarction area by Evans blue/TTC staining; maron staining detects the degree of myocardial fibrosis. The invention mainly uses animal experiments to clarify the cardiovascular protection effect of the class I Hdac6 small molecule agonist MDL-800 newly developed in the market.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to application of MDL-800 in myocardial ischemia reperfusion of mice.
Background
Myocardial infarction (myocardial infarction, MI) refers to ischemic necrosis of the myocardium, and on the basis of coronary artery lesions, the blood flow of the coronary artery is sharply reduced or interrupted, so that the corresponding myocardium is severely and continuously subjected to acute ischemia, and finally the ischemic necrosis of the myocardium is caused. Acute myocardial infarction is a serious health and life threatening disease for human beings, and within a few minutes after the onset of acute myocardial infarction, myocardial cells in the central region of ischemia die due to ischemia and hypoxia, and the global morbidity, disability rate and death rate are also high. However, how to improve myocardial infarction caused by myocardial ischemia is attracting extensive attention and research worldwide. The most effective current method for improving MI is to restore blood flow to the ischemic myocardium as soon as possible, i.e. ischemia reperfusion. And effective ischemia reperfusion can save about 30% of the patient's lives. Methods such as thrombolysis therapy, percutaneous transluminal coronary angioplasty, arterial bypass surgery, and extracorporeal circulation of cardiac surgery are generally used in clinic.
Myocardial ischemia/reperfusion injury (myocardial ischemia/reperfusion injury, MI/RI) refers to a condition where the myocardial blood supply is interrupted in a short period of time and the ischemic myocardium is damaged more seriously than before the blood supply is restored within a certain period of time after the blood supply is restored. It is generally thought that ischemic tissues and organs survive and recover from reperfusion before they are reversibly damaged, but it has been found that in some cases, the ischemic tissues and organs do not undergo significant changes in functional structure over time, but undergo irreversible damage when blood reperfusion is obtained. There are studies showing that reperfusion of cells that have developed severe ischemic injury does not reduce the injury, but rather accelerates cell death. In myocardial cell injury caused by ischemia, the metabolic rate of myocardial cells is obviously reduced, and tissues are damaged slowly but obviously. If oxygen is re-supplied on the basis of hypoxia, the damage is accelerated and aggravated. Although reperfusion re-supplies tissue cell oxygen and nutrients, this re-oxygenation instead accelerates body damage because oxygen radical release is promoted while re-oxygenation. Reperfusion plays an important role in a variety of pathological, physiological changes in the body, with reperfusion following ischemia leading to more serious consequences. Several factors have been found to play an important role in the mechanism of ischemia reperfusion injury, with reperfusion following ischemia leading to more serious consequences. Several factors have been found to play an important role in the mechanism of ischemia reperfusion injury, including inflammatory processes, oxygen radical damage, calcium overload, oxidative stress, and the like.
The prevention and treatment of myocardial ischemia/reperfusion injury has attracted great attention and widespread attention from doctors, scholars and researchers worldwide. Along with the wide development of vascular recanalization technologies such as percutaneous coronary stent implantation and coronary artery bypass grafting, the direct myocardial infarction rate is reduced, but reperfusion injury is correspondingly increased, and effective drug treatment is not yet available at present, and clinically ACEI/ARB drugs or beta receptor blockers can improve the prognosis of patients with myocardial infarction after vascular recanalization to a certain extent, but still cannot meet clinical requirements, and further development of drugs for improving the prognosis of patients with myocardial infarction after vascular recanalization is still the key point of current research. The prior basic research experiments show that the Hdac6 has low expression in various tumor tissues and proved to play an important role in tumor inhibition, so that the drug development aiming at the Hdac6 is continuously improved. MDL-800 is used as a newly developed class I Hdac6 small molecule agonist to increase Hdac 6-mediated histone H3 deacetylation, thereby inhibiting a plurality of adverse effects caused by histone H3 acetylation. Hdac6 plays an important role in the pathological process of various diseases, and the application of MDL-800 in preparing medicaments for inhibiting SARSCoV virus susceptibility is disclosed in a Chinese patent publication No. CN112972453A, which shows that MDL-800 can reduce the infection risk of SARS-CoV-2 virus. However, the role of the Hdac6 agonist MDL-800 in myocardial ischemia reperfusion injury has not been reported.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides an application of MDL-800 in myocardial ischemia reperfusion of mice.
The invention is realized in such a way that the application of MDL-800 in myocardial ischemia reperfusion of mice is realized by constructing a model of myocardial ischemia reperfusion of mice, injecting MDL-800 intraperitoneally 15min before reperfusion, detecting myocardial infarction areas of each group of mice by Evans blue/TTC staining, detecting myocardial scar fibers of each group of mice by Marsh staining, and verifying the effect of MDL-800 in myocardial ischemia reperfusion of mice, and comprises the following steps:
Step one, MDL-800 is a newly developed class I Hdac6 small molecule agonist in the market, and is purchased from the market, and animals adopt C57BL/6J mice;
step two, myocardial ischemia reperfusion animal model;
step three, injecting MDL-800 into the abdominal cavity 15min before recharging;
step four, the heart contraction function of the mice is detected by the small animals through ultrasonic;
fifthly, detecting myocardial infarction area by Evans blue/TTC staining;
and step six, carrying out myocardial scar fiber detection on each group of mice by using masson staining.
Further, the myocardial ischemia reperfusion animal model:
preoperative preparation: introducing mixed gas of isoflurane and oxygen into an animal preparation bin through a catheter by a Matrix VIP anesthesia machine, and performing induced inhalation anesthesia on each group of experimental mice; after the anesthesia is satisfied, fixing the mouse on an operating table, keeping the mouse at a constant temperature in a supine position, and connecting the mixed gas to the mouth and nose parts of the mouse through another catheter to maintain the anesthesia;
And (5) constructing a model.
Further, the model construction:
① Skin preparation in the operation area: for the mice subjected to ultrasonic detection after the operation, the skin preparation is carried out on the chest operation area of the mice before the operation, so that the operation incision cleavage and infection caused by the skin preparation after the operation are prevented;
② Partial chest opening: resolving the sternum position of a mouse, cutting an incision with the length of about 2-3cm by scissors at the position of 3-4mm near the heart at the left edge of the sternum position, enabling the incision direction to be perpendicular to the long axis of the heart, properly separating a chest wall structure by using hemostat, and making a discontinuous mattress type eversion suture opening around the incision by using a 4-0 suture line in advance;
③ Exposing the heart: after the chest wall is completely separated, the chest wall is quickly pierced by the hemostatic forceps, the chest incision is fully opened, the left-hand pressing and the right-hand rotating of the hemostatic forceps are coordinated, the heart is quickly extruded out of the chest, and the left anterior descending coronary artery is found;
④ Ligating the blood vessel: ligature the anterior descending branch of left coronary artery rapidly with 6-0 suture line, tie a slipknot, remove the suture line while facilitating reperfusion, avoid opening chest again; after ligation, the heart is quickly and carefully returned to the chest, and the chest wall is extruded to discharge the air in the chest as much as possible; rapidly tightening the reserved skin suture opening, ligating the incision, and closing the chest; care was taken to expose a portion of the slip knot at the ligation to the incision skin; group of sham operations: mice were operated in the same surgical group, except that the left anterior descending coronary artery was not ligated after chest opening to expose the heart.
Further, the small animal ultrasonically detects the heart contractile function of the mouse:
Preparation before detection: skin preparation is carried out before the ischemia reperfusion model is established, the chest of a mouse to be detected is enlarged and dehaired one day before ultrasonic detection, and an ultrasonic observation area is fully exposed;
Anesthesia: introducing mixed gas of isoflurane and oxygen into an animal preparation bin through a catheter by a Matrix VIP anesthesia machine, and performing induced inhalation anesthesia on each group of experimental mice; after anesthesia is satisfied, fixing the mice on a detection table by using an adhesive tape, and in a supine position, ensuring that limbs are on electrode plates of the detection table, simultaneously coating conductive adhesive, and maintaining the temperature of the detection table at 37-40 ℃; the mixed gas for anesthesia is connected to the mouth and nose parts of the mice through another catheter to maintain anesthesia; recording the physiological indexes of the electrocardio activity and the respiratory frequency of the mice, controlling the heart rate of the mice to be 400-500 times/min, and starting to perform the next step after stabilizing for a plurality of minutes;
Ultrasonic detection: after the chest of the mice is coated with ultrasonic medium glue, carrying out M-type ultrasonic detection on each group of mice by utilizing a Visual Sonics Vevo2100 type small animal ultrasonic detection system; the related parameters of the detection system are that the measurement frequency is 15MHz, the depth is 3cm, and the speed is 200mm/s; in order to obtain a relatively clear parasternal left ventricle short axis image, the direction and the position of the ultrasonic probe can be properly adjusted, and the corresponding M-type ultrasonic image is measured and recorded on the level of the papillary muscle of the left ventricle;
Statistical analysis: from the recorded M-mode ultrasound images, each group of mice Ejection Fraction (EF), fractional Shortening (FS), stroke Volume (SV), cardiac Output (CO) index was measured with ultrasound analysis software, and the detection required to include 6 consecutive cardiac cycles.
Further, the evans blue/TTC staining detects myocardial infarction area:
Preparing a dyeing solution: preparing 1% -3% Evan's blue solution and TTC solution by PBS solution, filtering to remove undissolved impurities, and pre-placing TTC solution in 37 deg.C water bath;
intravascular dye injection: injecting anaesthetized mice into the abdominal cavity by using sodium pentobarbital solution with the concentration of 0.3% 24 hours after constructing a myocardial ischemia reperfusion model, opening the thoracic cavity, exposing the heart, ligating the anterior descending branch of the left coronary artery again according to the original ligature position, sucking a proper amount of Evans blue solution by using an insulin injector, rapidly injecting a small amount of Evans blue dye from the root of the ascending aorta, stopping injection after the blood supply area of the heart is covered by the Evans blue dye, taking down the heart after the heart slightly beats, and placing the heart on dry ice;
Heart section: after the heart is frozen, cutting the heart into heart slices of 2mm from the apex of the heart by a blade, wherein the heart slices are generally cut into 5 slices, and the standard is that the Evan blue area accounts for 100%, 75%, 50%, 25% and 0% of the whole slice, and the heart slices are rapidly placed in TTC solution at 37 ℃ for 20min;
Taking a picture: taking out the heart slices after incubation is completed, placing the heart slices in PBS solution, and taking pictures by using a macro camera; 5-6 mice are selected from each group;
Picture analysis: each heart slice consists of a region of 2 or 3 colors, and the sham surgery group appears as 2 colors: blue, red; the surgical group appears in 3 colors: blue, red and white; myocardial infarction area was calculated using ImageJ image analysis software: risk area/total area of cardiac slice, white area/risk area.
Further, the masson stain detects the degree of myocardial fibrosis:
And (3) a Pinus massoniana dyeing step: ① Dewaxing a paraffin section of heart tissue to water; ② The cell nuclei are stained with Harris' hematoxylin for 10min and slightly washed with running water; ③ Differentiation of 0.1% hydrochloric acid alcohol, water washing, blue returning and water washing; ④ Masson composite dye liquor is dyed for 10-20min, and 0.2% acetic acid is quickly washed for 15 seconds twice; ⑤ 1% phosphomolybdic acid treatment for 58min,0.2% acetic acid quick washing for 15 seconds twice; ⑥ 1% bright green SF for 24min,0.2% acetic acid for 15 seconds twice; ⑦ Differentiating with fresh 95% alcohol for 20-30min; ⑧ After dehydration treatment by using absolute alcohol and transparency treatment by using dimethylbenzene, slicing and sealing heart paraffin by using neutral gum, and preserving at room temperature for later use;
The masson stained sections were observed: taking the stained sections of the heart masson of each group of mice was observed using a common optical microscope (Leica); blue or green (counterstained with benzene blue or bright green) as collagen fibers, red as cytoplasm, myofibers and erythrocytes, and blue brown as nuclei. Each group selected 5 mice.
In combination with the technical scheme and the technical problems to be solved, the technical scheme to be protected has the following advantages and positive effects:
Firstly, the invention mainly sets up myocardial ischemia reperfusion model to clarify the cardiovascular protection effect of the newly developed class I Hdac6 small molecule agonist MDL-800 in the market. MDL-800 is reported in literature to be a selective allosteric activator of SIRT6, SIRT6 is a member of SIRT deacetylase family and is responsible for deacetylation of histones H3K9Ac and H3K56Ac, and has tumor inhibition activity. MDL-800 increases the deacetylase activity of SIRT6 up to 22-fold, resulting in a decrease in H3K9Ac and H3K56Ac in hepatocellular carcinoma (HCC) cells, leading to cell cycle arrest and inhibition of proliferation thereof. It also inhibited the growth of the hepatocellular carcinoma cell line BEL7405 xenograft. However, the research in cardiovascular diseases has not been reported yet, and the specific action and mechanism thereof have not been clarified yet.
The invention discovers that MDL-800 obviously improves the heart contraction function after myocardial ischemia reperfusion, the invention constructs a mouse myocardial ischemia reperfusion injury model, the MDL-800 is injected intraperitoneally 15min before reperfusion (20 mg/kg, DMSO is dissolved, PEG400 is diluted by physiological saline), and an animal ultrasonic detection system is utilized to carry out M-type ultrasonic detection on each group of mice, so that compared with a sham operation group, the heart contraction function of the mice in the ischemia reperfusion group is reduced (FS, EF, SV, CO is reduced), and the heart function of the mice is improved (FS, EF, SV, CO is improved after MDL-800 is given).
The invention discovers that MDL-800 obviously improves myocardial infarction area after myocardial ischemia reperfusion, and for a mouse myocardial ischemia reperfusion injury model, MDL-800 is injected intraperitoneally 15min before reperfusion, myocardial infarction area detection is carried out on mice in each group through Evan blue/TTC staining, and the result shows that the myocardial infarction area of the mice in the ischemia reperfusion group is obviously increased, the myocardial infarction area is obviously reduced after MDL-800 is given, and the ischemia danger areas among 2 groups have no obvious difference.
The invention discovers that MDL-800 obviously improves myocardial scar fibers after myocardial ischemia reperfusion, and for a mouse myocardial ischemia reperfusion injury model, MDL-800 is injected into abdominal cavity 15min before reperfusion, myocardial scar fibers are detected on each group of mice by masson staining, and the result shows that the myocardial scar fibers of the mice in the ischemia reperfusion group are obviously increased, and the myocardial scar fibers are obviously lightened after MDL-800 is given.
Secondly, the invention has the technical effects and advantages that: (1) The application of MDL-800 in cardiovascular diseases has not been reported, the specific action and mechanism are not clear, the research of the invention has a certain exploring and innovative significance (2) MDL-800 as a small molecule agonist has been proved in the research of liver cancer, the application of MDL-800 in preventing and treating MI/IR has not been reported, and the invention has important research value and significance
Thirdly, the technical scheme of the invention fills the technical blank in the domestic and foreign industries: the technical scheme of the invention provides a new scheme for researching MI/IR, provides a new molecular agonist for treating and improving MI/IR, and supplements a new scheme for researching and treating MI/IR.
The technical scheme of the invention solves the technical problems that people are always desirous of solving but are not successful all the time: the technical scheme of the invention discovers a new role of MDL-800 in cardiovascular research from basic research, and provides a new thought for researching and treating technical problems of MI/IR.
Fourth, the significant technological advances made by the use of MDL-800 in mice myocardial ischemia reperfusion injury are mainly manifested in the following aspects:
1. Novel cardioprotection strategy
MDL-800 is used as a newly developed class I Hdac6 small molecule agonist, and provides a new treatment strategy for myocardial ischemia reperfusion injury. Compared with the traditional treatment means, MDL-800 directly acts on myocardial cells through a specific molecular mechanism to regulate the acetylation state of proteins, thereby protecting the heart from ischemia reperfusion injury.
2. Promotion of cardiac functional recovery
MDL-800 helps in the recovery of cardiac function by improving the survival rate of cardiomyocytes and alleviating reperfusion injury, especially in acute myocardial infarction and long-term myocardial ischemia reperfusion models. This technical advancement not only improves the therapeutic effect, but also reduces complications after heart disease, such as heart failure and myocardial fibrosis.
3. Reduction of myocardial infarction area
By using MDL-800, myocardial infarction area is significantly reduced, which means that the extent of heart damage is smaller and the structure and function of the heart remains more intact. This was verified in Evan blue/TTC staining and cardiac ultrasound testing, demonstrating the high efficacy of MDL-800 in cardiac protection.
4. Reduction of myocardial scar fibrosis
MDL-800 reduces scar fibrosis following myocardial ischemia reperfusion, which is critical to maintaining normal function of the heart. Scar fibrosis can lead to heart stiffness affecting its ability to pump blood, while the use of MDL-800 significantly reduces this risk, as demonstrated in masson staining.
5. Potential application of heart disease treatment
The successful application of MDL-800 in the mouse myocardial ischemia reperfusion injury model lays a foundation for the potential application thereof in the treatment of human heart diseases, in particular myocardial infarction and ischemic heart diseases. Future clinical studies further verify their safety and effectiveness, providing more treatment options for heart disease patients.
The application of MDL-800 in the myocardial ischemia reperfusion injury of mice provided by the invention shows remarkable technical progress and potential value in the aspects of heart protection and treatment of heart diseases.
Drawings
FIG. 1 is a flow chart of a method for applying MDL-800 to myocardial ischemia reperfusion injury of a mouse according to an embodiment of the present invention.
FIG. 2 is a graph showing the effect of MDL-800 on cardiac contractile function in mice with myocardial ischemia reperfusion, provided in an embodiment of the present invention. * P <0.05vs.Sham vs Vehicle groups; #p <0.05vs.IR vs Vehicle group.
FIG. 3 is a graph showing the effect of MDL-800 on myocardial infarction area in mice with myocardial ischemia reperfusion, provided in an embodiment of the present invention. #p <0.05vs.IR vs Vehicle group.
FIG. 4 is a graph showing the effect of MDL-800 on myocardial scar fibers in a myocardial ischemia reperfusion mouse, as provided by an embodiment of the present invention. * P <0.05vs.Sham vs Vehicle groups; #p <0.05vs.IR vs Vehicle group.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Application example 1: application of MDL-800 in acute myocardial infarction model
The effect of MDL-800 on recovery of cardiac function in mice after acute myocardial infarction was studied.
1. And (3) establishing a model: an acute myocardial infarction model was established on C57BL/6J mice by ligating the anterior descending left coronary artery to simulate the condition of human myocardial infarction.
2. And (3) drug intervention: MDL-800 was injected intraperitoneally immediately after ligation surgery and the dose was set to the optimal therapeutic dose determined by dose effect experiments prior to study.
3. Functional assessment: the heart function of mice was assessed using echocardiography on postoperative day 1, day 7, and day 14, with primary assessment metrics including Left Ventricular Ejection Fraction (LVEF) and left ventricular short axis shortening fraction (LVFS).
4. Histological analysis: at the end of the experiment, mice were dissected, hearts were taken for TTC staining to assess myocardial infarction area, and masson staining was used to assess the extent of myocardial fibrosis.
The MDL-800 treated mice were expected to have better recovery of cardiac function, less myocardial infarction area than the control group, and reduced myocardial fibrosis.
Application example 2: application of MDL-800 in long-term myocardial ischemia reperfusion model
The protective effect of MDL-800 on heart structure and function after long-term myocardial ischemia reperfusion injury was investigated.
1. And (3) establishing a model: myocardial ischemia reperfusion model was established on C57BL/6J mice by reperfusion after 30 minutes of temporary blocking of the left coronary artery.
2. And (3) drug intervention: MDL-800 was intraperitoneally injected 15 minutes prior to reperfusion, and the dose was determined according to pre-experiments.
3. Long-term observation: mice were observed for a long period of 8 weeks and heart structure and function were assessed periodically using small animal ultrasound.
4. Molecular mechanism research: after the experiment is finished, heart samples are collected for Western blot (Western blot) analysis, and the influence of MDL-800 on the expression of heart protection proteins, such as HSP70, bcl2 and the like, is studied.
5. Histological analysis: myocardial infarction area and myocardial fibrosis were assessed by evans blue/TTC staining and marsonian staining.
It is expected that better cardiac function protection, including higher LVEF and LVFS values, and reduced myocardial infarction area and degree of myocardial fibrosis, are observed in MDL-800 treated mice than in the control group.
These two examples demonstrate the use of MDL-800 in different myocardial ischemia reperfusion models and its potential cardioprotective effects, demonstrating the potential of MDL-800 for use in the field of cardiac disease treatment through detailed experimental design and prospective research results.
As shown in fig. 1, the present invention provides a method for the use of MDL-800 in myocardial ischemia reperfusion injury in mice comprising the steps of:
S101, MDL-800 adopted is a newly developed class I Hdac6 small molecule agonist in the market, and is purchased from the market, and animals adopt C57BL/6J mice;
S102, myocardial ischemia reperfusion animal model;
s103, injecting MDL-800 into the abdominal cavity 15min before recharging;
s104, performing M-type ultrasonic detection on each group of mice by using a small animal ultrasonic detection system;
s105, detecting myocardial infarction area by Evans blue/TTC staining;
S106, carrying out myocardial scar fiber detection on each group of mice by using masson staining.
The application of MDL-800 in myocardial ischemia reperfusion injury of mice is based on the following working principle:
mechanism of action of class I Hdac6 small molecule agonists
MDL-800: MDL-800 is a newly developed class I small molecule agonist of Hdac6 (histone deacetylase 6). Hdac6 is mainly involved in the deacetylation process of intracellular proteins, which is critical for many biological functions in cells, including gene expression regulation, protein stability, and signaling. By activating Hdac6, MDL-800 can regulate the acetylation state of specific proteins, thereby affecting the protection mechanism of myocardial cells and relieving the damage caused by myocardial ischemia reperfusion.
2. Establishment of myocardial ischemia reperfusion model
And (3) establishing a model: myocardial ischemia reperfusion models were established surgically on C57BL/6J mice, typically by temporarily blocking and then restoring left coronary blood flow. The model can simulate the pathophysiological state of human after myocardial infarction and is used for researching myocardial protection strategies.
Intervention Effect of MDL-800
And (3) drug intervention: mice were intervened by intraperitoneal injection of MDL-800 15 minutes before ischemia ended and reperfusion began. MDL-800 acts to reduce myocardial cell damage during reperfusion, protect myocardial cells, and improve cardiac contractile function by activating Hdac6 and its downstream signaling pathways.
4. Myocardial injury and functional assessment
Functional assessment: the heart contractile function of mice treated with MDL-800 was examined using a small animal ultrasonic technique, and the protective effect thereof on heart function was evaluated.
Myocardial infarction area detection: by staining with evans blue/TTC (2, 3,5 triphenyltetrazolium chloride), living (red) and dead (undyed or white) myocardial tissue can be intuitively distinguished, thereby accurately measuring myocardial infarction area.
Myocardial scar fiber detection: myocardial tissue is stained using masson staining, myocardial scarring and fibrosis can be assessed, and the long-term effects of MDL-800 on myocardial protection are further validated.
5. Comprehensive analysis
The working principle is comprehensive: MDL-800 can regulate signal path in myocardial cell by activating Hdac6, and relieve oxidative stress and cell injury caused by ischemia reperfusion, thereby reducing myocardial infarction area, improving heart function, reducing myocardial fibrosis, and improving heart contractility. The series of protective effects are helpful for improving the survival rate of myocardial cells after ischemia reperfusion, and has potential application value for treating heart diseases.
Through the specific experimental steps and the explanation of the working principle, the application of MDL-800 in the myocardial ischemia reperfusion injury of mice shows the potential value and application prospect in the field of heart protection.
The invention provides a myocardial ischemia reperfusion animal model:
preoperative preparation: introducing mixed gas of isoflurane and oxygen into an animal preparation bin through a catheter by a Matrix VIP anesthesia machine, and performing induced inhalation anesthesia on each group of experimental mice; after the anesthesia is satisfied, fixing the mouse on an operating table, keeping the mouse at a constant temperature in a supine position, and connecting the mixed gas to the mouth and nose parts of the mouse through another catheter to maintain the anesthesia;
And (5) constructing a model.
The invention provides model construction:
① Skin preparation in the operation area: for the mice subjected to ultrasonic detection after the operation, the skin preparation is carried out on the chest operation area of the mice before the operation, so that the operation incision cleavage and infection caused by the skin preparation after the operation are prevented;
② Partial chest opening: resolving the sternum position of a mouse, cutting an incision with the length of about 2-3cm by scissors at the position of 3-4mm near the heart at the left edge of the sternum position, enabling the incision direction to be perpendicular to the long axis of the heart, properly separating a chest wall structure by using hemostat, and making a discontinuous mattress type eversion suture opening around the incision by using a 4-0 suture line in advance;
③ Exposing the heart: after the chest wall is completely separated, the chest wall is quickly pierced by the hemostatic forceps, the chest incision is fully opened, the left-hand pressing and the right-hand rotating of the hemostatic forceps are coordinated, the heart is quickly extruded out of the chest, and the left anterior descending coronary artery is found;
④ Ligating the blood vessel: ligature the anterior descending branch of left coronary artery rapidly with 6-0 suture line, tie a slipknot, remove the suture line while facilitating reperfusion, avoid opening chest again; after ligation, the heart is quickly and carefully returned to the chest, and the chest wall is extruded to discharge the air in the chest as much as possible; rapidly tightening the reserved skin suture opening, ligating the incision, and closing the chest; care was taken to expose a portion of the slip knot at the ligation to the incision skin; group of sham operations: mice were operated in the same surgical group, except that the left anterior descending coronary artery was not ligated after chest opening to expose the heart.
The invention provides a small animal ultrasonic detection mouse heart contraction function:
Preparation before detection: skin preparation is carried out before the ischemia reperfusion model is established, the chest of a mouse to be detected is enlarged and dehaired one day before ultrasonic detection, and an ultrasonic observation area is fully exposed;
Anesthesia: introducing mixed gas of isoflurane and oxygen into an animal preparation bin through a catheter by a Matrix VIP anesthesia machine, and performing induced inhalation anesthesia on each group of experimental mice; after anesthesia is satisfied, fixing the mice on a detection table by using an adhesive tape, and in a supine position, ensuring that limbs are on electrode plates of the detection table, simultaneously coating conductive adhesive, and maintaining the temperature of the detection table at 37-40 ℃; the mixed gas for anesthesia is connected to the mouth and nose parts of the mice through another catheter to maintain anesthesia; recording the physiological indexes of the electrocardio activity and the respiratory frequency of the mice, controlling the heart rate of the mice to be 400-500 times/min, and starting to perform the next step after stabilizing for a plurality of minutes;
Ultrasonic detection: after the chest of the mice is coated with ultrasonic medium glue, carrying out M-type ultrasonic detection on each group of mice by utilizing a Visual Sonics Vevo2100 type small animal ultrasonic detection system; the related parameters of the detection system are that the measurement frequency is 15MHz, the depth is 3cm, and the speed is 200mm/s; in order to obtain a relatively clear parasternal left ventricle short axis image, the direction and the position of the ultrasonic probe can be properly adjusted, and the corresponding M-type ultrasonic image is measured and recorded on the level of the papillary muscle of the left ventricle;
Statistical analysis: from the recorded M-mode ultrasound images, each group of mice Ejection Fraction (EF), fractional Shortening (FS), stroke Volume (SV), cardiac Output (CO) index was measured with ultrasound analysis software, and the detection required to include 6 consecutive cardiac cycles.
The Evansi blue/TTC staining provided by the invention detects myocardial infarction area:
Preparing a dyeing solution: preparing 1% -3% Evan's blue solution and TTC solution by PBS solution, filtering to remove undissolved impurities, and pre-placing TTC solution in 37 deg.C water bath;
intravascular dye injection: injecting anaesthetized mice into the abdominal cavity by using sodium pentobarbital solution with the concentration of 0.3% 24 hours after constructing a myocardial ischemia reperfusion model, opening the thoracic cavity, exposing the heart, ligating the anterior descending branch of the left coronary artery again according to the original ligature position, sucking a proper amount of Evans blue solution by using an insulin injector, rapidly injecting a small amount of Evans blue dye from the root of the ascending aorta, stopping injection after the blood supply area of the heart is covered by the Evans blue dye, taking down the heart after the heart slightly beats, and placing the heart on dry ice;
Heart section: after the heart is frozen, cutting the heart into heart slices of 2mm from the apex of the heart by a blade, wherein the heart slices are generally cut into 5 slices, and the standard is that the Evan blue area accounts for 100%, 75%, 50%, 25% and 0% of the whole slice, and the heart slices are rapidly placed in TTC solution at 37 ℃ for 20min;
Taking a picture: taking out the heart slices after incubation is completed, placing the heart slices in PBS solution, and taking pictures by using a macro camera; 5-6 mice are selected from each group;
Picture analysis: each heart slice consists of a region of 2 or 3 colors, and the sham surgery group appears as 2 colors: blue, red; the surgical group appears in 3 colors: blue, red and white; myocardial infarction area was calculated using ImageJ image analysis software: risk area/total area of cardiac slice, white area/risk area.
Further, the masson stain detects the degree of myocardial fibrosis:
And (3) a Pinus massoniana dyeing step: ① Paraffin sections of heart tissue were dewaxed to water. ② Harris' hematoxylin stained nuclei for 10min and slightly washed with running water. ③ Differentiation of 0.1% hydrochloric acid alcohol, water washing, blue returning and water washing. ④ Masson composite dye liquor is dyed for 10-20min, and 0.2% acetic acid is quickly washed for 15 seconds twice. ⑤ 1% phosphomolybdic acid treatment is carried out for 5-8min, and 0.2% acetic acid is rapidly washed for 15 seconds twice. ⑥ 1% bright green SF for 2-4min and 0.2% acetic acid for 15 seconds. ⑦ Directly differentiating with fresh 95% alcohol for 20-30min. ⑧ After dehydration treatment with absolute alcohol and transparency treatment with xylene, heart paraffin sections were sealed with neutral gum and stored at room temperature for use.
The masson stained sections were observed: the uptake of the heart masson stained sections of each group of mice was observed using a common light microscope (Leica). Blue or green (counterstained with benzene blue or bright green) as collagen fibers, red as cytoplasm, myofibers and erythrocytes, and blue brown as nuclei. Each group selected 5 mice.
The invention mainly uses animal experiments to clarify the cardiovascular protection effect of the class I Hdac6 small molecule agonist MDL-800 newly developed in the market.
Application example 1: according to the invention, by constructing a myocardial ischemia reperfusion injury model of mice, injecting MDL-800 (20 mg/kg, DMSO is dissolved, PEG400 is diluted by physiological saline) into abdominal cavity 15min before reperfusion, carrying out M-type ultrasonic detection on each group of mice by using a small animal ultrasonic detection system, measuring indexes such as Ejection Fraction (EF), fractional Shortening (FS), stroke Volume (SV), cardiac Output (CO) of each group of mice by using ultrasonic analysis software, and the detection needs to comprise 6 continuous cardiac cycles. The results showed that the ischemia reperfusion group mice had reduced cardiac contractile function (FS, EF, SV, CO decreased in both), and the mice had improved cardiac function (FS, EF, SV, CO increased in both) after MDL-800 administration, as compared to the sham group. The invention discovers that MDL-800 obviously improves the heart contraction function after myocardial ischemia reperfusion.
Application example 2: according to the invention, by constructing a myocardial ischemia reperfusion injury model of mice, injecting MDL-800 into abdominal cavity 15min before reperfusion, detecting myocardial infarction areas of mice in each group by Evan blue/TTC staining, wherein each heart section consists of 2 or 3 color areas, the artificial operation group is 2 colors (blue and red), and the operation group is 3 colors (blue, red and white). Myocardial infarction area (risk area/total heart slice area, white area/risk area) was calculated using Image J Image analysis software (version 1.8). The results show that the myocardial infarction area of the mice in the ischemia reperfusion group is obviously increased, the myocardial infarction area is obviously reduced after MDL-800 is given, and the ischemia danger areas among the 2 groups have no obvious difference, so that the invention discovers that the MDL-800 obviously improves the myocardial infarction area after myocardial ischemia reperfusion.
Application example 3: according to the invention, by constructing a mouse myocardial ischemia reperfusion injury model, injecting MDL-800 into the abdominal cavity 15min before reperfusion, detecting myocardial scar fibers of each group of mice by masson staining, and observing and taking heart masson stained sections of each group of mice by using a common optical microscope (Leica). Blue or green (counterstained with benzene blue or bright green) as collagen fibers, red as cytoplasm, myofibers and erythrocytes, and blue brown as nuclei. Each group selected 5 mice. The result shows that the myocardial scar fiber of the mice in the ischemia reperfusion group is obviously increased, and the myocardial scar fiber is obviously lightened after MDL-800 is given, and the invention discovers that the MDL-800 obviously improves the myocardial scar fiber after myocardial ischemia reperfusion.
The embodiment of the invention has a great advantage in the research and development or use process, and has the following description in combination with data, charts and the like of the test process.
As shown in FIG. 2, the invention finds that MDL-800 significantly improves the heart contractile function after myocardial ischemia reperfusion, and the result shows that compared with a sham operation group, the heart contractile function of a mouse in the ischemia reperfusion group is reduced (FS, EF, SV, CO is reduced), and the heart function of the mouse is improved (FS, EF, SV, CO is improved) after MDL-800 is given.
As shown in figure 3, the invention discovers that MDL-800 obviously improves myocardial infarction area after myocardial ischemia reperfusion, and the result shows that the myocardial infarction area of mice in an ischemia reperfusion group is obviously increased, the myocardial infarction area is obviously reduced after MDL-800 is given, and the ischemia danger areas among 2 groups have no obvious difference.
As shown in figure 4, the invention finds that MDL-800 obviously improves myocardial scar fibers after myocardial ischemia reperfusion, and the result shows that myocardial scar fibers of mice in an ischemia reperfusion group are obviously increased, and the myocardial scar fibers are obviously relieved after MDL-800 is given.
The invention mainly uses animal experiments to clarify the cardiovascular protection effect of the class I Hdac6 small molecule agonist MDL-800 newly developed in the market.
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.
Claims (7)
1. The application of MDL-800 in myocardial ischemia reperfusion injury of mice is characterized in that the MDL-800 is injected intraperitoneally 15min before reperfusion by constructing a model of myocardial ischemia reperfusion of the mice, myocardial infarction area detection is carried out on each group of mice by Evan blue/TTC staining, and the effect of the MDL-800 in myocardial ischemia reperfusion of the mice is verified.
2. The use of MDL-800 according to claim 1, in a myocardial ischemia reperfusion injury in a mouse, comprising the steps of:
Step one, MDL-800 is a newly developed class I Hdac6 small molecule agonist in the market, and is purchased from the market, and animals adopt C57BL/6J mice;
step two, myocardial ischemia reperfusion animal model;
step three, injecting MDL-800 into the abdominal cavity 15min before recharging;
step four, the heart contraction function of the mice is detected by the small animals through ultrasonic;
fifthly, detecting myocardial infarction area by Evans blue/TTC staining;
and step six, carrying out myocardial scar fiber detection on each group of mice by using masson staining.
3. The use of MDL-800 according to claim 2, wherein the myocardial ischemia reperfusion animal model:
Preoperative preparation: introducing mixed gas of isoflurane and oxygen into an animal preparation bin through a catheter by a Matrix VIP anesthesia machine, and performing induced inhalation anesthesia on each group of experimental mice; after the anesthesia is satisfied, the mice are fixed on an operating table, kept at a constant temperature in a supine position, and the mixed gas is connected to the oral and nasal parts of the mice through another catheter to maintain the anesthesia.
4. The use of MDL-800 according to claim 3, wherein the model is constructed by:
① Skin preparation in the operation area: for the mice subjected to ultrasonic detection after the operation, the skin preparation is carried out on the chest operation area of the mice before the operation, so that the operation incision cleavage and infection caused by the skin preparation after the operation are prevented;
② Partial chest opening: resolving the sternum position of a mouse, cutting an incision with the length of about 2-3cm by scissors at the position of 3-4mm near the heart at the left edge of the sternum position, enabling the incision direction to be perpendicular to the long axis of the heart, properly separating a chest wall structure by using hemostat, and making a discontinuous mattress type eversion suture opening around the incision by using a 4-0 suture line in advance;
③ Exposing the heart: after the chest wall is completely separated, the chest wall is quickly pierced by the hemostatic forceps, the chest incision is fully opened, the left-hand pressing and the right-hand rotating of the hemostatic forceps are coordinated, the heart is quickly extruded out of the chest, and the left anterior descending coronary artery is found;
④ Ligating the blood vessel: ligature the anterior descending branch of left coronary artery rapidly with 6-0 suture line, tie a slipknot, remove the suture line while facilitating reperfusion, avoid opening chest again; after ligation, the heart is quickly and carefully returned to the chest, and the chest wall is extruded to discharge the air in the chest as much as possible; rapidly tightening the reserved skin suture opening, ligating the incision, and closing the chest; care was taken to expose a portion of the slip knot at the ligation to the incision skin; group of sham operations: mice were operated in the same surgical group, except that the left anterior descending coronary artery was not ligated after chest opening to expose the heart.
5. The use of MDL-800 according to claim 2, wherein the small animal ultrasonically detects the contractile function of the mouse heart in a mouse myocardial ischemia reperfusion injury:
Preparation before detection: skin preparation is carried out before the ischemia reperfusion model is established, the chest of a mouse to be detected is enlarged and dehaired one day before ultrasonic detection, and an ultrasonic observation area is fully exposed;
Anesthesia: introducing mixed gas of isoflurane and oxygen into an animal preparation bin through a catheter by a Matrix VIP anesthesia machine, and performing induced inhalation anesthesia on each group of experimental mice; after anesthesia is satisfied, fixing the mice on a detection table by using an adhesive tape, and in a supine position, ensuring that limbs are on electrode plates of the detection table, simultaneously coating conductive adhesive, and maintaining the temperature of the detection table at 37-40 ℃; the mixed gas for anesthesia is connected to the mouth and nose parts of the mice through another catheter to maintain anesthesia; recording the physiological indexes of the electrocardio activity and the respiratory frequency of the mice, controlling the heart rate of the mice to be 400-500 times/min, and starting to perform the next step after stabilizing for a plurality of minutes;
Ultrasonic detection: after the chest of the mice is coated with ultrasonic medium glue, carrying out M-type ultrasonic detection on each group of mice by utilizing a Visual Sonics Vevo2100 type small animal ultrasonic detection system; the related parameters of the detection system are that the measurement frequency is 15MHz, the depth is 3cm, and the speed is 200mm/s; in order to obtain a relatively clear parasternal left ventricle short axis image, the direction and the position of the ultrasonic probe can be properly adjusted, and the corresponding M-type ultrasonic image is measured and recorded on the level of the papillary muscle of the left ventricle;
Statistical analysis: based on the recorded M-mode ultrasound images, the indices of each group of mice EF, FS, SV, CO were measured using ultrasound analysis software, and the test required to include 6 consecutive cardiac cycles.
6. The use of MDL-800 according to claim 2, wherein the evans blue/TTC staining detects myocardial infarction area:
Preparing a dyeing solution: preparing 1% -3% Evan's blue solution and TTC solution by PBS solution, filtering to remove undissolved impurities, and pre-placing TTC solution in 37 deg.C water bath;
intravascular dye injection: injecting anaesthetized mice into the abdominal cavity by using sodium pentobarbital solution with the concentration of 0.3% 24 hours after constructing a myocardial ischemia reperfusion model, opening the thoracic cavity, exposing the heart, ligating the anterior descending branch of the left coronary artery again according to the original ligature position, sucking a proper amount of Evans blue solution by using an insulin injector, rapidly injecting a small amount of Evans blue dye from the root of the ascending aorta, stopping injection after the blood supply area of the heart is covered by the Evans blue dye, taking down the heart after the heart slightly beats, and placing the heart on dry ice;
Heart section: after the heart is frozen, cutting the heart into heart slices of 2mm from the apex of the heart by a blade, wherein the heart slices are generally cut into 5 slices, and the standard is that the Evan blue area accounts for 100%, 75%, 50%, 25% and 0% of the whole slice, and the heart slices are rapidly placed in TTC solution at 37 ℃ for 20min;
Taking a picture: taking out the heart slices after incubation is completed, placing the heart slices in PBS solution, and taking pictures by using a macro camera; 5-6 mice are selected from each group;
Picture analysis: each heart slice consists of a region of 2 or 3 colors, and the sham surgery group appears as 2 colors: blue, red; the surgical group appears in 3 colors: blue, red and white; myocardial infarction area was calculated using ImageJ image analysis software: risk area/total area of cardiac slice, white area/risk area.
7. The use of MDL-800 according to claim 2, wherein the masson staining detects the degree of myocardial fibrosis in a mouse myocardial ischemia reperfusion injury:
And (3) a Pinus massoniana dyeing step: ① Dewaxing a paraffin section of heart tissue to water; ② The cell nuclei are stained with Harris' hematoxylin for 10min and slightly washed with running water; ③ Differentiation of 0.1% hydrochloric acid alcohol, water washing, blue returning and water washing; ④ Masson composite dye liquor is dyed for 10-20min, and 0.2% acetic acid is quickly washed for 15 seconds twice; ⑤ 1% phosphomolybdic acid treatment is carried out for 5-8min, and 0.2% acetic acid is rapidly washed for 15 seconds twice; ⑥ Dyeing with 1% bright green SF for 2-4min, and washing with 0.2% acetic acid for 15 seconds twice; ⑦ Differentiating with fresh 95% alcohol for 20-30min; ⑧ After dehydration treatment by using absolute alcohol and transparency treatment by using dimethylbenzene, slicing and sealing heart paraffin by using neutral gum, and preserving at room temperature for later use;
the masson stained sections were observed: applying a common optical microscope; taking the heart masson stained sections of each group of mice; blue or green is collagen fiber, red is cytoplasmic, myofiber and erythrocyte, blue brown is nucleus. Each group selected 5 mice.
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