Use of 2-methylenebutyrolactone in the preparation of a medicament for the treatment of myocardial ischemia / reperfusion injury

Abstract

The application relates to the technical field of medicines, and particularly discloses an application of 2-methenylbutyrolactone in preparation of a medicine for resisting myocardial ischemia / reperfusion injury. A small-molecule compound 2-methenylbutyrolactone (Tulipalin A, TA) capable of activating glutathione synthesis and having good protection effect is found. Researches of the application find 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, reduce the heart injury of mice and improve the heart function, and is further developed into a medicine for resisting MI / R injury.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of medicines, in particular to application of 2-methylene butyrolactone in preparation of a medicine for resisting myocardial ischemia / reperfusion injury. BACKGROUND

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

[0003] Although some drugs are currently used in clinical treatment of MI / R injury, the therapeutic effect of these drugs is 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

[0004] The application aims to overcome the shortcomings of the prior art and provide application of 2-methylene butyrolactone in preparation of a medicine for resisting myocardial ischemia / reperfusion injury.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the application is as follows:

[0006] The application provides application of 2-methylene butyrolactone in preparation of a medicine for resisting myocardial ischemia / reperfusion injury.

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

[0008]

[0009] The application screens out a small molecule compound Tulipalin A (TA) that can activate glutathione synthesis and has good protection effect by constructing an in vitro high-throughput screening model of MI / R (myocardial ischemia / reperfusion injury). The application 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, reduce mouse heart injury and improve heart function, and is further developed into a medicine for resisting MI / R injury.

[0010] As a preferred embodiment of the application, the mass concentration of the 2-methylenyl butyrolactone is 3-15 μM.

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

[0012] Experiments show that adding 2-methylenyl butyrolactone can increase the number of myocardial living cells, and shows an obvious dose-increasing effect.

[0013] The application also provides an application of 2-methylenyl butyrolactone in preparing a preparation for protecting myocardial disease after hypoxia / reoxygenation injury.

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

[0015] As a preferred embodiment of the application, the myocardial disease includes non-familial restrictive cardiomyopathy.

[0016] In a non-familial restrictive cardiomyopathy hypoxia / reoxygenation injury model, adding 2-methylenyl butyrolactone can effectively improve the levels of intracellular GSH and GSSG.

[0017] It is shown that adding 2-methylenyl butyrolactone can up-regulate the glutathione level and has a protective effect on non-familial restrictive cardiomyopathy after hypoxia / reoxygenation injury.

[0018] As a preferred embodiment of the application, the 2-methylenyl butyrolactone up-regulates the glutathione level.

[0019] The application also provides an application of 2-methylenyl butyrolactone in preparing a drug for treating functional injury of human engineered heart tissue caused by hypoxia / reoxygenation.

[0020] As a preferred embodiment of the application, the 2-methylenyl butyrolactone improves cell death, oxidative stress level and contraction function of human engineered heart tissue caused by hypoxia / reoxygenation.

[0021] Experiments show that adding 2-methylenyl butyrolactone can obviously reduce fibrosis of human engineered heart tissue caused by hypoxia / reoxygenation, and shows an obvious dose-decreasing effect. And adding 2-methylenyl butyrolactone can obviously reduce functional injury of human engineered heart tissue caused by hypoxia / reoxygenation.

[0022] As a preferred embodiment of the application, the medicine further comprises a pharmaceutically acceptable carrier.

[0023] The application also provides an anti-myocardial ischemia / reperfusion injury medicine, which comprises 2-methylenyl butyrolactone and a pharmaceutically acceptable carrier.

[0024] Through animal experiments, it is found that the left ventricular ejection fraction (LVEF) of the mouse heart after myocardial ischemia / reperfusion is significantly reduced, and the left ventricular fractional shortening (LVFS) is greatly reduced; and the heart function of the 2-methylenyl butyrolactone protective medicine group is significantly recovered.

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

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

[0027] The application provides an application of 2-methylenyl butyrolactone in the preparation of an anti-myocardial ischemia / reperfusion injury medicine, and finds a small molecule compound 2-methylenyl butyrolactone (Tulipalin A, TA) which can activate glutathione synthesis and has good protection effect. The application 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, reduce the injury of the mouse heart and improve the heart function, and is further developed into an anti-MI / R injury medicine. Moreover, the addition of 2-methylenyl butyrolactone can obviously reduce the fibrosis of human engineering heart tissue caused by hypoxia / reoxygenation, and has an obvious dose-decreasing effect. Moreover, the addition of 2-methylenyl butyrolactone can obviously reduce the functional injury of human engineering heart tissue caused by hypoxia / reoxygenation. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The figure is the result graph of the influence of 2-methylenyl butyrolactone on the cell death, oxidative stress level and cell structure of myocardial cells after MI / R injury in Example 1.

[0029] Figure 2 The figure is the result graph of the influence of 2-methylenyl butyrolactone on the cell death, oxidative stress level and contraction function of human heart engineering tissue after MI / R injury in Example 2.

[0030] Figure 3 The figure is the result graph of the influence of 2-methylenyl butyrolactone on the heart function and infarct area of the mouse after MI / R injury in Example 3. DETAILED DESCRIPTION

[0031] For better illustrating the purpose, technical scheme and advantages of the present application, the present application will be further described in conjunction with the drawings and specific examples.

[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 commercially available unless otherwise specified, and the component raw materials used in each parallel experiment are the same.

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

[0034] Example 1

[0035] Experimental material: neonatal rat myocardial cells.

[0036] The primary neonatal rat cells used are derived from the heart of 1-3 day old (Sprague Dawley, SD) rats, which are isolated by trypsin and collagenase digestion, and high-purity (≥98%) primary rat myocardial cells are obtained after differential adhesion.

[0037] Experimental method:

[0038] Primary rat neonatal myocardial cell isolation: after disinfecting the neonatal rats (5) with 75% alcohol, the left hand pinches the back skin of the neonatal rat, the sternum is cut open from the left margin of the xiphoid with scissors, the heart is exposed and taken out with tweezers and washed in PBS, and the washed heart is placed in another glass dish to remove as much connective tissue and atrium as possible; the heart is cut into pieces with scissors, 5 mL of digestion solution (2.5 mL of 0.25% trypsin + 2.5 mL of DMEM / F12 + 5 μg of collagenase type II) is added, and then it is transferred to a graduated cylinder and placed in a 37°C water bath for digestion. The first digestion is for 30 minutes, the supernatant after digestion is collected and transferred to a centrifuge tube containing 20 mL of DMEM containing 10% FBS, 5 mL of digestion solution is added and the myocardial cells are dispersed gently, and the digestion is continued in the water bath; the supernatant is collected every 20 minutes thereafter for 3 times; the collected digestion solution is filtered through a 100 μm filter and centrifuged at room temperature at 2000 rpm / min for 5 minutes; the supernatant is discarded, an appropriate amount of DMEM containing 10% FBS is added, and the cells are cultured by adhesion for 60 minutes, the culture dish is removed, shaken gently several times, the suspension containing myocardial cells is transferred to a 50 mL centrifuge tube, centrifuged at 2000 rpm / min for 5 minutes, and the supernatant is discarded, and the cells are resuspended with myocardial cell culture medium.

[0039] Establishment of in vitro high-throughput screening model: the digested neonatal rat myocardial cells were resuspended to 1×10 4Cells were seeded at a density of 1 cell / well into gelatin-coated 384-well plates. After 36 hours of culture, cells were washed with PBS, and glucose-free serum-free DMEM medium was added to place cells in a 0.1% oxygen concentration three-gas incubator for hypoxia for 12 hours. After hypoxia, fresh DMEM / F12 cell culture medium was added to place cells in a culture incubator for recovery of normal oxygen concentration (21%) for 12 hours.

[0040] Based on the above myocardial cell hypoxia / reoxygenation injury model, 8402 different small molecule compounds in the small molecule compound library were screened to finally obtain the best protective effect compound Tulipalin A. Tulipalin A small molecule compound was added for treatment under two administration modes (addition of drug during hypoxia and reoxygenation: H+R; addition of drug only during reoxygenation: R), and Tulipalin A was divided into a low-dose (3 μM) group and a high-dose (10 μM) group.

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

[0042] Calcein / PI / Hoechst staining of cells: Calcein-AM (calcein), PI (propidium iodide) and Hoechst were added to DMEM basic medium for cell staining, and the working solution concentrations were 2 μM, 4.5 μM and 1 μg / ml, respectively. The prepared staining working solution was added to the cells treated with small molecule compounds, and incubated at 37°C for 15 minutes. High-content imaging analysis system was used to detect Calcein / PI / Hochst fluorescence and imaging.

[0043] TUNEL staining: After fixation of myocardial cells with 4% paraformaldehyde, green fluorescently labeled myocardial cell specific troponin cTnT (myocardial cell marker) was used, and then staining was performed according to the operation steps of TUNEL cell apoptosis detection kit (Vazyme Biotech, A113-02). High-content imaging analysis system was used to detect cTNT / TUNEL / DAPI fluorescence and imaging.

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

[0045] GSH and GSSG detection: The operation was performed according to the GSH and GSSG detection kit (Biyun, S0053) steps, and the enzyme-labeled instrument was used for detection.

[0046] Results, as shown in Figure 1

[0047] Figure 1 -A and Figure 1 -B are the Calcein / PI / Hoechst staining results and statistical column charts of each treatment group, in which Calcein (green, live cells), PI (orange, dead cells), and Hoechst (blue, cell nuclei). The results show that, compared with the normal group, the number of surviving cells in the H / R+DMSO treatment group is significantly reduced, and the proportion of living cells in the Tulipalin A protective drug group is significantly higher than that in the H / R+DMSO group, showing a clear dose-dependent effect. The experiment proves that the Tulipalin A small molecule compound can significantly reduce the death of myocardial cells caused by hypoxia / reoxygenation and promote cell survival.

[0048] Figure 1 -C and Figure 1 -D are the TUNEL staining results and apoptosis cell proportion statistical charts of each treatment group. Among them, TUNEL (red, apoptotic cells), cTnT (green, myocardial cells), and Hoechst (blue, cell nuclei). The results show that the number of apoptotic cells in the H / R+DMSO treatment group is significantly increased compared with the normoxic group, and the number of apoptotic cells in the Tulipalin A small molecule compound treatment group is significantly reduced compared with the H / R+DMSO treatment group. The experiment proves that the Tulipalin A small molecule compound can significantly reduce the apoptosis rate of myocardial cells after hypoxia / reoxygenation.

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

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

[0051] The above experiments collectively demonstrate that the candidate drug Tulipalin A in high-throughput drug screening can up-regulate the level of glutathione and has a protective effect on NRCM after hypoxia / reoxygenation injury.

[0052] Example 2

[0053] Experimental materials: human engineered heart tissue. The human myocardial cells used are derived from myocardial cells differentiated from human embryonic stem cells, and the human embryonic stem cells are from WiCell Institute in Wisconsin, USA.

[0054] Experimental method:

[0055] Human engineered heart tissue construction: the human embryonic stem cell differentiated myocardial cells are digested into single cells and counted; a PDMS mold is prepared for EHT molding; a nylon fiber paper frame is used to support the EHT; a specimen needle is used to fix the paper frame; the PDMS mold is immersed in 75% alcohol, ultrasonically cleaned (20 min, 2 times), and high-temperature sterilized together with the paper frame and specimen needle, and then dried and taken to the intercellular space for use; the mold is placed, washed with sterilized water twice, completely dried, and then 0.2% F127 is added dropwise on the surface of the mold until the surface is completely covered without bubbles, and then left to stand for more than 1 h; the F127 is absorbed, the mold is washed with sterilized water twice, and then DPBS is used to wash the mold once, the paper frame is placed on the mold, the specimen needle is used to fix the four corners, and DPBS is added dropwise in the mold groove for soaking; the prepared myocardial cells are added to the mold groove at a concentration of 1.0-1.5 x 10 6Cells were resuspended in 1x medium at a concentration of 1.5 x 105cells / 60 μl; 2.4 μl of 50 U / mL thrombin was added to each 60 μl of cell suspension to make A solution; A solution was aliquoted into EP tubes at 120 μl / tube and stored until use; one EP tube was placed on ice to prepare 120 μl of B solution: 48 μl of 2x medium + 48 μl of fibrinogen + 24 μl of Matrigel; the liquid in the prepared mold was completely aspirated and stored until use; 120 μl of B solution was quickly added to 120 μl of A solution to make 240 μl of EHT gel solution, which was added to the PDMS mold in two times, 120 μl each time; the gun head was preheated for 5-10 s by holding it with the fingers, and then the gel was vertically spotted into the PDMS mold (if the gel coagulates quickly, the preheating time can be shortened or omitted); after the gel was spotted, the EHT gel and mold were placed in a 37°C incubator for 10-30 min to allow the gel to completely coagulate; the sample needle was removed, and the EHT was taken out of the mold by pulling the paper frame and placed in the medium for culture (if the EHT is in a long strip shape and cannot be immediately taken out, it is placed in the medium together with the mold for 12 h before being taken out); for EHT culture, the first day can use basal medium + 10% FBS + 20 μM cytarabine + 10 μM Y27632, and then use early stage medium, and after one week, use late stage medium.

[0056] The present application uses the EHT model to explore the hypoxia / reoxygenation injury condition, and finally determines that the oxygen concentration is 1%, the hypoxia time is 8 h, and the reoxygenation time is 48 h.

[0057] Based on the above myocardial cell hypoxia / reoxygenation injury model, Tulipalin A small molecule compounds were added under two administration modes (H+R: adding drugs during both hypoxia and reoxygenation; R: adding drugs only during reoxygenation), and Tulipalin A was divided into 5 μM dose group and 15 μM dose group.

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

[0059] Masson staining: take out the frozen section and dry at room temperature, wash with PBS for 3 times, 3 min each time. After washing the embedding agent, draw a hydrophobic circle around the tissue section with an immunohistochemical pen. All subsequent steps are operated within the hydrophobic circle; add appropriate amount of 4% paraformaldehyde to fix the tissue section at room temperature for 20-30 min; put the section into the medium dyeing solution and dye at room temperature for one night or in a 57-60℃ incubator for 1 h, and then wash with running water for 10 min; drop dye with azurite blue staining solution for 5 min, wash with water for 2 times, 10-15 s each time; drop dye with Mayer hematoxylin staining solution for 5 min, wash with distilled water for 2 times, 10-15 s each time; differentiate with acid ethanol differentiation solution for a few seconds until the tissue turns completely red, wash with water to stop differentiation, and wash with distilled water for 10 min; drop dye with ponceau red staining solution for 3 min, wash with distilled water for 2 times, 10-15 s each time; treat with phosphomolybdate solution for 10 min; pour off the solution, do not wash the section with water, and directly drop with aniline blue staining solution for 1 min; drop with weak acid working solution to cover the section for 2 min; dehydrate with 95% ethanol for 30 s; dehydrate with anhydrous ethanol for 2 times, 30 s for the first time and 1 min for the second time; clear with xylene for 2 times, 1-2 min each time; and seal with neutral gum.

[0060] Electric field video analysis: open the stepping motor, electric stimulation device, temperature control device, signal converter, signal amplifier and corresponding software; level and zero the mechanical sensor, and add Perfusion buffer in the detection tank; cut an EHT, and fix the two ends of the nylon frame on the mechanical sensor with specimen needles; after the EHT is contracted stably, collect the mechanical signal of the EHT for 30 s when it spontaneously contracts; apply a constant voltage at a rated frequency to continuously stimulate and collect the mechanical signal of the EHT for 30 s; use the stepping motor to stretch the EHT, and collect the mechanical signal for 30 s each time, with a constant stretching length of 2% of the original length; after the EHT is stretched to 12%, stop the electric stimulation, and collect the mechanical signal for 30 s, and then restore it to the original length.

[0061] The results are shown in Table 1. Figure 2

[0062] Figure 2 -A and Figure 2 -B are the Masson staining results and statistical column charts of each treatment group, in which red represents myocardial cells and blue represents fibroblasts. The results show that, compared with the normal group, the fibrosis level of the H / R+DMSO treatment group is significantly increased, while the fibrosis level of the Tulipalin A protective drug group is significantly lower than that of the H / R+DMSO group, and presents an obvious dose-decreasing effect. The experiment proves that the 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 histogram and time statistical histogram of reaching 50% peak value of the response of each treatment group to electric field stimulation.

[0064] The results show that: compared with the normal group, the amplitude of the H / R+DMSO treatment group is significantly reduced, and the time to reach the peak value is longer, while the amplitude of the Tulipalin A protective drug group is significantly higher than that of the H / R+DMSO group, and the time to reach the peak value is significantly restored, and presents a significant dose-dependent effect. Experiments prove that Tulipalin A small molecule compound 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 method:

[0068] Establishment of mouse ischemia / reperfusion model: 8-10 week-old, 18-25 g male C57BL / 6J mice were randomly divided into sham operation group (Sham group, only thoracotomy and threading operation without ligation); ischemia / reperfusion injury+DMSO group (I / R+DMSO group, ischemia for 45 min, remove the ligation for 2 min, intraperitoneal injection of DMSO solution after reperfusion for 24 h); ischemia / reperfusion injury+Tulipalin A group (I / R+Tulipalin A group, ischemia for 45 min, remove the ligation for 2 min, intraperitoneal injection of 5 mg / kg, 10 mg / kg, 15 mg / kg TA solution after reperfusion for 24 h).

[0069] After reperfusion for 24 h, the survival rate of mice was more than 90%, and the success rate of myocardial ischemia / reperfusion modeling was more than 80%. After ligation, it can be obviously found that the blood supply of the left ventricular part below the ligation line is blocked and presents grayish white. After reperfusion for 24 h, cardiac function was detected by echocardiography, and infarct size was detected by TTC / Evans blue staining.

[0070] Small animal echocardiography: After the mice were anesthetized with 1-1.5% isoflurane, they were fixed on the echocardiography detection platform, and the hair on the chest of the mice was removed with 2% isoflurane continuous anesthesia or a hair removal blade. The hot table was turned on, and the temperature was controlled at 40°C. The mice with the chest hair removed were placed on the operation table with the abdomen facing up. Pure water (for electrical conduction) was applied to the limbs and fixed to the corresponding position of the operation table with adhesive tape. The operation table was adjusted so that the head of the mouse was slightly higher than the tail, and the apex and bottom of the heart of the mouse were flush. Two pieces of thick adhesive film were placed on both sides of the chest of the mouse (fixed conductive adhesive), and ultrasonic coupling agent was applied to the chest of the mouse, about 2 cm thick. The probe should be buried in the ultrasonic coupling agent during measurement. The Vevo 3100 400-MHz high-resolution small animal ultrasonic real-time imaging system was used for transthoracic echocardiography. The left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and other cardiac function indicators were recorded synchronously.

[0071] TTC / Evans blue staining: Prepare 5% Evans blue with normal saline or PBS, and prepare 1% TTC with adult cell perfusion fluid. Anesthetize the mice, insert the respirator, open the chest, and re-ligate the same position of the ischemia / reperfusion operation. Inject 3-4 ml of 5% Evans blue into the superior vena cava of the mouse. Remove the heart, wipe it clean with paper, and wash it in a 6 cm dish with PBS or normal saline. Place the washed heart on an ice block, and together with the ice block, place it in a -80°C freezer for 3-5 min. Slice the ligation line to the apex, and cut 4-6 pieces of the same thickness. Immerse the slices in 1% TTC staining solution at 37°C for 20 min. After completion, remove the myocardial slices, rinse them with PBS for a while, arrange them flat, and fix them in a 4% paraformaldehyde solution. Take photos using a high-power upright fluorescence microscope, and both the front and back of the slice need to be photographed.

[0072] The results are shown in Figure 3 .

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

[0074] Figure 3- D to Figure 3 - F is the result of TTC / Evans blue staining of each treatment. The results show that Tulipalin A can significantly reduce the infarct size of the heart of mice after ischemia / reperfusion, and reduce ischemia / reperfusion injury.

[0075] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not a limitation on the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present application.

Claims

1. Use of 2-methylenebutyrolactone for the preparation of a medicament for the treatment of myocardial ischemia / reperfusion injury, characterized in that, The myocardial ischemia / reperfusion injury is a specific injury caused by oxidative stress and apoptosis during the reperfusion process after myocardial ischemia; the mass concentration of the 2-methylenbutyrolactone is 3-15 μM; The 2-methylenbutyrolactone reduces myocardial cell death, ROS accumulation, oxidative stress level and cell structure disorder caused by myocardial ischemia / reperfusion.

2. Use of 2-methylenbutyrolactone in preparation of a preparation for protecting myocardial disease after hypoxia / reoxygenation injury.

3. Use according to claim 2, wherein the compound is ###0002### The myocardial disease includes non-familial restrictive cardiomyopathy.

4. The use according to claim 2, wherein the compound is ###0002### The 2-methylenbutyrolactone up-regulates glutathione level.

5. Use of 2-methylenbutyrolactone in preparation of a drug for treating functional injury of human engineered heart tissue caused by hypoxia / reoxygenation.

6. The use according to claim 5, wherein the compound is ###0002### The 2-methylenbutyrolactone improves cell death, oxidative stress level and contraction function of human engineered heart tissue caused by hypoxia / reoxygenation.

7. The use according to claim 1, wherein The drug further comprises a pharmaceutically acceptable carrier.

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