Application of 2-methylene butyrolactone in preparation of medicine for resisting myocardial ischemia / reperfusion injury

Abstract

The invention relates to the technical field of medicines, and particularly discloses application of 2-methylene butyrolactone in preparation of a medicine for resisting myocardial ischemia / reperfusion injury. The invention discloses a small molecule compound, namely, 2-methylene butyrolactone (Tulipalin A, TA), which can activate synthesis of glutathione and has a good protection effect. Research finds that Tulipalin A can reduce myocardial cell death, ROS accumulation and structural disorder caused by ischemia / reperfusion, improve the contraction function of human heart engineering tissue, relieve mouse heart injury and improve the heart function, and the Tulipalin A is further researched and developed into an anti-MI / R injury medicine.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and in particular to an application of 2-methylbutyrolactone in the preparation of a drug for preventing myocardial ischemia / reperfusion injury. Background Art

[0002] Cardiovascular disease (CVD) refers to a class of diseases that affect the heart and blood vessels. Common risk factors include high blood pressure, high cholesterol, smoking, diabetes, obesity, lack of exercise and unhealthy diet. Cardiovascular disease may cause myocardial infarction. After myocardial infarction, dredging blood vessels is the primary treatment strategy, but reperfusion therapy can cause myocardial ischemia / reperfusion (MI / R) injury, leading to further damage to myocardial cells. Its complex pathological mechanism and high mortality rate make its treatment a focus and difficulty in medical research.

[0003] Although there are some drugs currently used in the clinic to treat MI / R injury, the therapeutic effects of these drugs are not ideal. Therefore, developing new drugs and treatment strategies to more comprehensively intervene in the pathological process of MI / R injury is an important direction of current research. Summary of the invention

[0004] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and provide an application of 2-methylbutyrolactone in the preparation of a drug for preventing myocardial ischemia / reperfusion injury.

[0005] To achieve the above purpose, the technical solution adopted by this application is:

[0006] The present application provides the use of 2-methenylbutyrolactone in the preparation of a drug for preventing myocardial ischemia / reperfusion injury.

[0007] Among them, Tulipalin A (α-methylene-γ-butyrolactone) is a glycoside derived from tulip bulbs, CAS: 547-65-9, Chinese alias 2-methylenylbutyrolactone, a sesquiterpene lactone, is considered to be a cyclic analogue of the most common vinyl monomer methyl methacrylate, and its molecular structure is shown in formula (I):

[0008]

[0009] This application screened out Tulipalin A (TA), a small molecule compound that can activate glutathione synthesis and has good protective effects by constructing an in vitro high-throughput screening model of MI / R (myocardial ischemia / reperfusion injury). This application found that Tulipalin A can reduce myocardial cell death, ROS accumulation, and structural disorder caused by ischemia / reperfusion, improve the contractile function of human cardiac engineering tissue, reduce cardiac damage in mice, and improve cardiac function, and further research and develop it into a drug against MI / R injury.

[0010] As a preferred embodiment of the application described in the present application, the mass concentration of the 2-methylenylbutyrolactone is 3 to 15 μM.

[0011] As a preferred embodiment of the application described in the present application, the 2-methylbutyrolactone reduces myocardial cell death, ROS accumulation, oxidative stress level and cell structure disorder caused by myocardial ischemia / reperfusion.

[0012] Experiments have shown that the addition of 2-methylbutyrolactone can increase the number of viable cardiomyocytes and show an obvious dose-escalation effect.

[0013] The present application also provides the use of 2-methylbutyrolactone in the preparation of a preparation for protecting cardiomyopathy after hypoxia / reoxygenation injury.

[0014] In the technical solution of the present application, the use of 2-methylbutyrolactone can significantly reduce myocardial cell death caused by hypoxia / reoxygenation, promote cell survival, and significantly reduce the apoptosis rate of myocardial cells after hypoxia / reoxygenation, and significantly reduce the oxidative stress level of myocardial cells after hypoxia / reoxygenation.

[0015] As a preferred embodiment of the application described in the present application, the cardiomyopathy includes non-familial restrictive cardiomyopathy.

[0016] In the hypoxia / reoxygenation injury model of non-familial restrictive cardiomyopathy, the addition of 2-methylbutyrolactone can effectively increase the levels of intracellular GSH and GSSG.

[0017] It was shown that the addition of 2-methylbutyrolactone could upregulate glutathione levels and have a protective effect on non-familial restrictive cardiomyopathy after hypoxia / reoxygenation injury.

[0018] As a preferred embodiment of the application described in the present application, the 2-methylenylbutyrolactone upregulates glutathione levels.

[0019] The present application also provides the use of 2-methenylbutyrolactone in the preparation of a drug for treating functional damage of human engineered heart tissue caused by hypoxia / reoxygenation.

[0020] As a preferred embodiment of the application described in the present application, the 2-methylenylbutyrolactone improves cell death, oxidative stress level and contractile function of human engineered heart tissue caused by hypoxia / reoxygenation.

[0021] Experiments have shown that the addition of 2-methylbutyrolactone can significantly reduce the fibrosis of human engineered heart tissue caused by hypoxia / reoxygenation, and shows an obvious dose-decreasing effect. In addition, the addition of 2-methylbutyrolactone can significantly reduce the functional damage of human engineered heart tissue caused by hypoxia / reoxygenation.

[0022] As a preferred embodiment of the application described in the present application, the drug further includes a pharmaceutically acceptable carrier.

[0023] The present application also provides a drug for preventing myocardial ischemia / reperfusion injury, which comprises 2-methylbutyrolactone and a pharmaceutically acceptable carrier.

[0024] Animal experiments have shown that after myocardial ischemia / reperfusion, the left ventricular ejection fraction (LVEF) of mice was significantly reduced, and the left ventricular fractional shortening (LVFS) was greatly reduced; while the heart function of the group receiving the protective drug 2-methylbutyrolactone was significantly restored.

[0025] Moreover, the addition of 2-methylbutyrolactone can significantly reduce the infarct area of ​​the mouse heart after myocardial ischemia / reperfusion and alleviate ischemia / reperfusion injury.

[0026] Compared with the prior art, this application has the following beneficial effects:

[0027] The present application provides the use of 2-methenylbutyrolactone in the preparation of drugs against myocardial ischemia / reperfusion injury. The present application has discovered a small molecule compound 2-methenylbutyrolactone (Tulipalin A, TA) that can activate glutathione synthesis and has a good protective effect. The present application has found that Tulipalin A can reduce myocardial cell death, ROS accumulation, and structural disorder caused by ischemia / reperfusion, improve the contractile function of human cardiac engineering tissue, reduce cardiac damage in mice and improve cardiac function, and further research and develop it into a drug against MI / R injury. In addition, the addition of 2-methenylbutyrolactone can significantly reduce the fibrosis of human engineered heart tissue caused by hypoxia / reoxygenation, and shows a significant dose-decreasing effect. In addition, the addition of 2-methenylbutyrolactone can significantly reduce the functional damage of human engineered heart tissue caused by hypoxia / reoxygenation. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The figure is a result of the effect of 2-methenylbutyrolactone on cell viability, oxidative stress level and cell structure of myocardial cells injured by MI / R in Example 1;

[0029] Figure 2 This is a graph showing the effect of 2-methenylbutyrolactone on cell survival, oxidative stress level and contractile function of human cardiac engineering tissue after MI / R injury in Example 2;

[0030] Figure 3 This is a graph showing the effect of 2-methenylbutyrolactone on cardiac function and infarct size in mice after MI / R injury in Example 3. DETAILED DESCRIPTION

[0031] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0032] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are of the same type.

[0033] Among them, I / R stands for ischemia / reperfusion, and H / R stands for hypoxia / reoxygenation.

[0034] Example 1

[0035] Experimental Materials: Neonatal rat cardiomyocytes.

[0036] The primary neonatal rat cells used were derived from the hearts of 1-3 day old (Sprague Dawley, SD) rats. They were digested and separated with trypsin and collagenase, and high-purity (≥98%) primary rat cardiomyocytes were obtained after differential adhesion.

[0037] Experimental methods:

[0038] Isolation of primary rat neonatal cardiomyocytes: After disinfecting neonatal rats with 75% alcohol (5 rats), pinch the skin on the back of the neonatal rats with the left hand, cut the sternum from the left edge of the xiphoid process with scissors, expose the heart, take it out with forceps, and wash it in PBS. Place the washed heart in another glass dish, and remove the connective tissue and atrium as much as possible; cut the heart into pieces with scissors, add 5 mL of digestion solution (2.5 ml 0.25% trypsin + 2.5 ml DMEM / F12 + 5 μg type II collagenase), and then transfer it to a graduated bottle with a pipette, and place it in a 37°C water bath for digestion. The first digestion was carried out for 30 minutes, and the supernatant after digestion was collected and transferred to a centrifuge tube containing 20 mL of DMEM containing 10% FBS. Then 5 mL of digestion solution was added and the cardiomyocytes were gently dispersed, and digestion was continued in a water bath; the supernatant was collected once every 20 minutes, and repeated 3 times; the collected digestion solution was passed through a 100 μm filter and centrifuged at 2000 rpm / min at room temperature for 5 minutes; the supernatant was discarded, and an appropriate amount of DMEM containing 10% FBS was added, and the cells were cultured for 60 minutes. The culture dish was removed and gently shaken several times, and the suspension containing the cardiomyocytes was transferred to a 50 mL centrifuge tube, centrifuged at 2000 rpm / min for 5 minutes, the supernatant was discarded, and the cells were resuspended in cardiomyocyte culture medium.

[0039] In vitro high-throughput screening model was established: digested neonatal rat cardiomyocytes were cultured at 1×10 4The cells were inoculated into a gelatin-coated 384-well plate at a density of 10 cells / well. After 36 hours of culture, the cells were washed with PBS, and glucose-free and serum-free DMEM medium was added. The cells were placed in a three-gas incubator with an oxygen concentration of 0.1% for hypoxia for 12 hours. After hypoxia, fresh DMEM / F12 cell culture medium was added and the cells were placed in an incubator to restore normal oxygen concentration (21%) for 12 hours.

[0040] Based on the above-mentioned myocardial cell hypoxia / reoxygenation injury model, 8402 different small molecule compounds in the small molecule compound library were screened and finally the compound with the best protective effect, Tulipalin A, was obtained. Tulipalin A small molecule compound was added under two dosing methods (drugs were added during both hypoxia and reoxygenation: H+R; drugs were added only during reoxygenation: R), and Tulipalin A was divided into a low-dose (3μM) group and a high-dose (10μM) group.

[0041] The experimental groups and culture conditions are as follows: 1. Normoxia group; 2. H / R (hypoxia / reoxygenation) + DMSO group; 3. (H+R) + Tulipalin A (3μM) group; 4. (H+R) + Tulipalin A (10μM) group; 5. R + Tulipalin A (3μM) group; 6. R + Tulipalin A (10μM) group; each group has 10 replicate wells.

[0042] Cell Calcein / PI / Hoechst staining: Calcein-AM (calcein), PI (propidium iodide) and Hoechst were added to DMEM basal culture medium for cell staining at the same time, and the working solution concentrations were 2μM, 4.5μM and 1μg / ml respectively. The prepared staining working solution was added to the cells treated with small molecule compounds, incubated at 37°C for 15 minutes, and Calcein / PI / Hochst fluorescence was detected and imaged using a high-content imaging analysis system.

[0043] TUNEL staining: cardiomyocytes were fixed with 4% paraformaldehyde, and then labeled with green fluorescence to express cardiomyocyte-specific troponin cTnT (cardiomyocyte marker), followed by staining according to the TUNEL apoptosis detection kit (Vazyme Biotech, A113-02). High-content imaging analysis system was used to detect cTNT / TUNEL / DAPI fluorescence and image.

[0044] ROS staining: DCFH-DA was diluted with DMEM / F12 medium at 1:1000 to a final concentration of 10 μM. The cell culture medium was removed, and the diluted DCFH-DA was added to cover the cells. The cells were incubated in a 37°C cell culture incubator for 20 min. The cells were washed three times with DPBS. The cTNT / TUNEL / DAPI fluorescence was detected and imaged using a high-content imaging analysis system.

[0045] GSH and GSSG detection: follow the steps of GSH and GSSG detection kit (Biyuntian, S0053) and use enzyme reader for detection.

[0046] Results, such as Figure 1 shown.

[0047] Figure 1 -A and Figure 1 -B is the Calcein / PI / Hoechst staining result and statistical bar graph of each treatment group, including Calcein (green, live cells), PI (orange, dead cells), and Hoechst (blue, cell nucleus). The results showed that compared with the normal group, the number of surviving cells in the H / R+DMSO treatment group was significantly reduced, while the proportion of live cells in the group with Tulipalin A protection drug was significantly higher than that in the H / R+DMSO group, and showed an obvious dose-increasing effect. Experiments have shown that Tulipalin A small molecule compound can significantly reduce myocardial cell death caused by hypoxia / reoxygenation and promote cell survival.

[0048] Figure 1 -C and Figure 1 -D is a representative graph of TUNEL staining results and a statistical graph of apoptotic cell ratios in each treatment group. Among them, TUNEL (red, apoptotic cells), cTnT (green, cardiomyocytes), and Hoechst (blue, cell nuclei). The results showed that the number of apoptotic cells in the H / R+DMSO treatment group increased significantly compared with the normoxia group, and the number of apoptotic cells in the Tulipalin A small molecule compound treatment group was significantly reduced compared with the H / R+DMSO treatment group. Experiments have shown that Tulipalin A small molecule compound can significantly reduce the apoptosis rate of cardiomyocytes after hypoxia / reoxygenation.

[0049] Figure 1 -E and Figure 1 -F is a representative picture of DCFH-DA staining results and fluorescence intensity statistical graphs of each treatment group. Among them, DCFH-DA (green, reactive oxygen species), Hoechst (blue, cell nucleus). By comparing the DCFH-DA fluorescence intensity of each group, it is proved that Tulipalin A small molecule compound can significantly reduce the oxidative stress level of cardiomyocytes after hypoxia / reoxygenation.

[0050] like Figure 1 -G and Figure 1 -H, Tulipalin A can effectively increase the levels of intracellular GSH and GSSG in the NRCM hypoxia / reoxygenation injury model.

[0051] The above experiments collectively demonstrate that Tulipalin A, a candidate drug for high-throughput drug screening, can upregulate glutathione levels and has a protective effect on NRCMs after hypoxia / reoxygenation injury.

[0052] Example 2

[0053] Experimental materials: Human engineered heart tissue. The human cardiomyocytes used were derived from cardiomyocytes differentiated from human embryonic stem cells, which were obtained from the WiCell Institute in Wisconsin, USA.

[0054] Experimental methods:

[0055] Construction of human engineered cardiac tissue: Digest the cardiomyocytes differentiated from human embryonic stem cells into single cells and count them; prepare a PDMS mold for EHT molding; a nylon fiber paper frame for supporting EHT; a specimen pin for fixing the paper frame; immerse the PDMS mold in 75% alcohol, ultrasonically clean (20 min, twice), sterilize it together with the paper frame and specimen pin at high temperature, and bring it into the cell space after drying for use; place the mold, wash it twice with sterile water, and after completely drying, drip 0.2% F127 on the surface of the mold until the surface is completely covered without bubbles, and let it stand for more than 1 hour; absorb F127, wash the mold twice with sterile water, and wash the mold once with DPBS, place the paper frame on the mold, fix the four corners with specimen pins, and drip DPBS in the mold groove for infiltration for use; place the prepared cardiomyocytes at a rate of 1.0-1.5×10 6Resuspend the cells / 60μl in 1× culture medium; add 2.4μl 50U / mL thrombin to each 60μl cell suspension to form solution A; dispense solution A into EP tubes at 120μl / tube for later use; take an EP tube and place it on ice to prepare 120μl solution B: 48μl 2× culture medium + 48μl fibrinogen + 24μl matrix gel; completely drain the liquid in the prepared mold for later use; take 120μl solution B and quickly add it to 120μl solution A and mix well to form 240μl Take 120μl of EHT gel solution twice each time, hold the gun tip with your fingers to preheat for 5-10s, then vertically spot the gel into the PDMS mold (if the gel coagulates quickly, the preheating time can be shortened or omitted); after the gel is dispensed, place the EHT gel and the mold in a 37°C incubator for 10-30min to allow the gel to completely coagulate; remove the specimen needle, pull the paper frame to remove the EHT from the mold, and place it in culture medium for culture (if the EHT is in a long strip shape and is not convenient to remove immediately, place it in the culture medium together with the mold and culture it for 12h before taking it out); for EHT culture, basal culture medium + 10% FBS + 20μM arabinoside + 10μM Y27632 can be used on the first day, then change to early culture medium, and change to late culture medium after one week.

[0056] The present application used the EHT model to explore the conditions of hypoxia / reoxygenation injury, and ultimately determined that the oxygen concentration was 1%, the hypoxia time was 8 hours, and the reoxygenation time was 48 hours.

[0057] Based on the above-mentioned myocardial cell hypoxia / reoxygenation injury model, Tulipalin A small molecule compound was added under two administration methods (drug was added during both hypoxia and reoxygenation: H+R; drug was added only during reoxygenation: R). Tulipalin A was divided into 5μM dosage group and 15μM dosage group.

[0058] The experimental groups and culture conditions are as follows: 1. Normoxia group; 2. H / R (hypoxia / reoxygenation) + DMSO group; 3. (H+R) + Tulipalin A (5μM) group; 4. (H+R) + Tulipalin A (15μM) group; 5. R + Tulipalin A (5μM) group; 6. R + Tulipalin A (15μM) group; each group has 10 replicate wells.

[0059] Masson staining: Take out the frozen sections and dry them at room temperature, then wash them three times with PBS, 3 minutes each time. After washing off the embedding agent, use an immunohistochemistry pen to draw a hydrophobic circle around the tissue section. All subsequent steps are performed within the hydrophobic circle; add an appropriate amount of 4% paraformaldehyde to fix the tissue sections at room temperature for 20 to 30 minutes; put the sections into the mordant solution, let it act at room temperature overnight or place them in a 57-60°C incubator for 1 hour for mordant staining, then rinse with running water for 10 minutes; drip stain with lapis lazuli blue staining solution for 5 minutes, wash with water twice, 10 to 15 seconds each time; drip stain with Mayer hematoxylin staining solution for 5 minutes, wash with distilled water twice, 10 to 15 seconds each time; differentiate with acidic ethanol differentiation solution for a few seconds until the tissue turns completely red , wash with water to terminate differentiation, rinse with distilled water for 10 minutes; stain with Ponceau fuchsin staining solution for 3 minutes, wash with distilled water twice, 10-15 seconds each time; treat with phosphomolybdic acid solution for 10 minutes; pour off the upper liquid, do not wash the slices with water, and directly add aniline blue staining solution for 1 minute; add weak acid working solution to cover the slices for 2 minutes; dehydrate with 95% ethanol for 30 seconds; dehydrate with anhydrous ethanol twice, 30 seconds for the first time and 1 minute for the second time; transparentize with xylene twice, 1-2 minutes each time; seal with neutral gum.

[0060] Electric field video analysis: turn on the stepper motor, electrical stimulation device, temperature control device, signal converter, signal amplifier and corresponding software; level and zero the mechanical sensor, and add Perfusion buffer to the detection tank; cut a piece of EHT, and fix the two ends of the nylon frame on the mechanical sensor with specimen pins; collect the mechanical signal of EHT spontaneous contraction for 30 seconds after the EHT contraction stabilizes; apply constant voltage and rated frequency for continuous electrical stimulation and collect the EHT mechanical signal for 30 seconds; use a stepper motor to stretch the EHT, and each stretching length is constant at 2% of the original length, and collect the mechanical signal for 30 seconds; after the EHT is stretched to 12%, stop the electrical stimulation, collect the mechanical signal for 30 seconds, and then restore it to its original length.

[0061] The results are as follows Figure 2 shown.

[0062] Figure 2 -A and Figure 2 -B is the Masson staining result and statistical bar graph of each treatment group, where red represents cardiomyocytes and blue represents fibroblasts. The results showed that compared with the normal group, the fibrosis level of the H / R+DMSO treatment group was significantly increased, while the fibrosis level of the Tulipalin A protective drug group was significantly lower than that of the H / R+DMSO group, and showed an obvious dose-decreasing effect. Experiments have shown that Tulipalin A small molecule compound can significantly reduce the fibrosis of human engineered heart tissue caused by hypoxia / reoxygenation.

[0063] Figure 2 -C to Figure 2-E is the waveform diagram, amplitude statistical bar graph and time statistical bar graph of reaching 50% peak value of each treatment group in response to electric field stimulation.

[0064] The results showed that compared with the normal group, the amplitude of the H / R+DMSO treatment group was significantly reduced and the time to reach the peak was prolonged, while the amplitude of the group with the Tulipalin A protective drug was significantly higher than that of the H / R+DMSO group and the time to reach the peak was significantly restored, showing a significant dose-dependent effect. The experiment proved that the small molecule compound Tulipalin A can significantly reduce the functional damage of human engineered heart tissue caused by hypoxia / reoxygenation.

[0065] Example 3

[0066] Experimental materials: 8-10 week old male C57BL / 6 mice, all selected mice were purchased from the Experimental Animal Center of Sun Yat-sen University.

[0067] Experimental methods:

[0068] Establishment of mouse cardiac ischemia / reperfusion model: Male C57BL / 6J mice aged 8-10 weeks and weighing 18-25 g were randomly divided into sham operation group (Sham group, only thoracotomy and threading were performed without ligation); ischemia / reperfusion injury + DMSO group (I / R+DMSO group, ischemia for 45 min, ligation was removed for 2 min, DMSO solution was intraperitoneally injected, and then reperfusion was performed for 24 h); ischemia / reperfusion injury + Tulipalin A group (I / R+Tulipalin A group, ischemia for 45 min, ligation was removed for 2 min, 5 mg / kg, 10 mg / kg, 15 mg / kg of TA solution was intraperitoneally injected, and then reperfusion was performed for 24 h).

[0069] After 24 hours of reperfusion, the survival rate of mice was over 90%, and the success rate of myocardial ischemia / reperfusion modeling was over 80%. After ligation, it was clearly found that the blood supply to the left ventricle below the ligature was blocked and appeared grayish white. After 24 hours of reperfusion, cardiac ultrasound was performed to detect cardiac function and TTC / Evans blue staining was performed to detect infarct area.

[0070] Small animal echocardiography: After mice were anesthetized with 1-1.5% isoflurane, they were fixed on the echocardiography platform, and 2% isoflurane was used for continuous anesthesia. The hair on the chest of the mice was removed with a depilatory or a razor blade. The heating stage was turned on and the temperature was controlled at 40°C. The mice with the chest hair removed were placed on the operating table with their abdomen facing up. Pure water (for conductivity) was applied to the limbs and fixed to the corresponding position on the operating table with tape. The operating table was adjusted so that the head of the mouse was slightly higher than the tail, so that the apex and bottom of the mouse heart were flush. Two thick films were placed on both sides of the mouse chest (to fix the conductive glue), and the ultrasound coupling agent was applied to the mouse chest with a thickness of about 2 cm. The probe should be buried in the ultrasound coupling agent during measurement. Transthoracic echocardiography was performed using the Vevo 3100 400-MHz high-resolution small animal ultrasound real-time imaging system. Cardiac function indicators such as left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were recorded simultaneously.

[0071] TTC / Evans blue staining: 5% Evans blue is prepared with saline or PBS, and 1% TTC is prepared with adult cell perfusion solution; anesthetize the mouse, put on a ventilator, open the chest and re-ligate the same position of the ischemia / reperfusion surgery; inject 3-4ml 5% Evans blue into the superior vena cava of the mouse; remove the heart, wipe it with paper, and wash it in PBS or saline in a 6cm dish; put the washed heart on ice and put it in a -80℃ refrigerator for 3-5min; slice from the ligature to the apex of the heart, cut 4-6 slices of the same thickness, and immerse the slices in 1% TTC staining solution at 37℃ for 20min. After completion, remove the myocardial slices, rinse with PBS for a while, flatten them, and fix them in 4% paraformaldehyde solution; use an advanced upright fluorescence microscope to take pictures, and both the front and back of the slices need to be taken.

[0072] The results are as follows Figure 3 shown.

[0073] Figure 3 -A to Figure 3 -C is the ultrasound results of each treatment. The results show that compared with the normal group, the left ventricular ejection fraction (LVEF) of the mouse heart was significantly reduced after ischemia / reperfusion, and the left ventricular fractional shortening (LVFS) was greatly reduced. The heart function of the group with the protection drug Tulipalin A was significantly restored.

[0074] Figure 3-D to Figure 3 -F are the results of TTC / Evans blue staining of each treatment. The results showed that Tulipalin A can significantly reduce the infarct area of ​​the heart after ischemia / reperfusion in mice and alleviate ischemia / reperfusion injury.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

Application of 1.2-methenylbutyrolactone in the preparation of drugs against myocardial ischemia / reperfusion injury.

2. The use according to claim 1, characterized in that The mass concentration of the 2-methenylbutyrolactone is 3 to 15 μM.

3. The use according to claim 1, characterized in that The 2-methylbutyrolactone reduces myocardial cell death, ROS accumulation, oxidative stress level and cell structure disorder caused by myocardial ischemia / reperfusion.

4. Use of 2-methenylbutyrolactone in the preparation of a preparation for protecting cardiomyopathy after hypoxia / reoxygenation injury.

5. The use according to claim 4, characterized in that The cardiomyopathy includes non-familial restrictive cardiomyopathy.

6. The use according to claim 4, characterized in that The 2-methylbenzylbutyrolactone upregulates glutathione levels.

7. Application of 2-methenylbutyrolactone in the preparation of drugs for treating functional damage of human engineered heart tissue caused by hypoxia / reoxygenation.

8. The use according to claim 7, characterized in that The 2-methylenylbutyrolactone improves cell death, oxidative stress level and contractile function of human engineered heart tissue caused by hypoxia / reoxygenation.

9. The use according to claim 1, characterized in that The medicament further includes a pharmaceutically acceptable carrier.

10. A drug for preventing myocardial ischemia / reperfusion injury, characterized in that: The drug comprises 2-methylbutyrolactone and a pharmaceutically acceptable carrier.

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