Use of trichinella cysteine protease inhibitors in acute myocardial infarction and subsequent ventricular remodeling

Abstract

This invention discloses the application of a Trichinella spiralis cysteine ​​protease inhibitor in acute myocardial infarction and subsequent ventricular remodeling, belonging to the field of medicinal chemistry. This invention provides a drug that can prevent, alleviate, and / or treat acute myocardial infarction and subsequent ventricular remodeling. The active ingredient of this drug is the Trichinella spiralis cysteine ​​protease inhibitor cystatin, abbreviated as rTs-Cys. rTs-Cys can effectively delay acute myocardial infarction and inflammatory response in mice, reduce the infarct area, improve cardiac function deterioration, and reverse organ pathological damage, providing a new approach for the clinical treatment of acute myocardial infarction and subsequent ventricular remodeling.

Description

Technical Field

[0001] This invention relates to the application of Trichinella spiralis cysteine ​​protease inhibitors in acute myocardial infarction and subsequent ventricular remodeling, and belongs to the field of medicinal chemistry technology. Background Technology

[0002] Myocardial infarction (MI) is a disease caused by a sudden reduction or interruption of coronary blood flow, leading to severe and persistent ischemia and hypoxia in the corresponding myocardium, ultimately resulting in ischemic necrosis of the myocardium based on a non-infectious immune response. With changing lifestyles and an aging population, cardiovascular diseases, especially congestive heart failure and malignant arrhythmias caused by myocardial infarction, have extremely high morbidity and mortality rates worldwide, seriously threatening human health. In recent years, with the widespread use of thrombolysis, interventional cardiac surgery, and the rapid development of drug therapy in MI treatment, the prognosis of MI patients has greatly improved. However, many patients still fail to undergo timely revascularization for various reasons, resulting in irreversible myocardial death, ventricular remodeling (VR), deterioration of cardiac function, and ultimately, heart failure. The occurrence of ventricular remodeling depends not only on the size of the infarct area but also on the infiltration of inflammatory cells and the repair of the infarcted myocardium. Therefore, effectively delaying the inflammatory response after MI and reversing excessive ventricular remodeling is crucial.

[0003] Based on the "hygiene hypothesis," "worm therapy," a strategy using worms and their derivatives to prevent and treat various diseases such as immune metabolic and inflammatory diseases, has received widespread attention. Cystatins secreted by worms are a class of highly effective inhibitors of cysteine ​​proteases. Based on amino acid sequence, the cysteine ​​protease inhibitor family is divided into Stefins (family 1), Cystatins (family 2), and Kininogens (family 3). Worm cystatins can inhibit cysteine ​​proteases, both suppressing the activity of tissue proteases in the host and regulating host cytokines to suppress the host's immune response. Experiments have shown that cystatins from filarial worms and Clonorchis sinensis improve acute colitis in mice; adoptive infusion of regulatory T cells induced by Cystatin from Echinochloa wiltii significantly reduces colitis in mice; and cystatins from Schistosoma japonicum can treat arthritis, sepsis, and colitis. The full-length cDNA sequence of Trichinella spiralis cystatin is numbered BQ692489.1 in the gene bank. The open reading frame of the full-length cDNA sequence is 588 bp, encoding 195 amino acids, with a theoretical molecular weight of 45 kDa. While reports have confirmed that Trichinella spiralis cystatin can treat sepsis and colitis, its ability to prevent, alleviate, or treat acute myocardial infarction and subsequent ventricular remodeling remains unexplored. Summary of the Invention

[0004] The purpose of this invention is to provide the application of rTs-Cys in acute myocardial infarction and subsequent ventricular remodeling. Animal experiments have demonstrated that rTs-Cys can delay acute myocardial infarction and inflammatory response in mice, reduce the infarct area, improve cardiac function deterioration, and reverse organ pathological damage, providing a new approach for the clinical treatment of acute myocardial infarction and subsequent ventricular remodeling.

[0005] Application of Cystatin, a Trichinella spiralis cysteine ​​protease inhibitor, in the preparation of drugs for the prevention, relief, and / or treatment of acute myocardial infarction and subsequent ventricular remodeling.

[0006] In one embodiment, the nucleotide sequence of the Cystatin is shown in SEQ ID NO.1.

[0007] In one embodiment, the prevention, mitigation, and / or treatment of acute myocardial infarction and subsequent ventricular remodeling includes at least one of the following:

[0008] (1) Reduce the mortality rate and cardiac index of acute myocardial infarction;

[0009] (2) Reduce the heart-to-body weight ratio and the area of ​​myocardial infarction;

[0010] (3) Reduce the degree of myocardial fibrosis after myocardial infarction and delay the damage to cardiac function;

[0011] (4) Inhibit the levels of pro-inflammatory cytokines and increase the expression of immune regulatory factors;

[0012] (5) Reduces inflammatory cell infiltration at the junction of myocardial infarction in MI mice;

[0013] (6) Reduce the level of cardiac troponin T in mice with acute myocardial infarction.

[0014] In one embodiment, Cystatin is used alone as the active ingredient of the drug.

[0015] In one embodiment, the effective dose of Cystatin is 0.75–1.5 mg / kg.

[0016] In one embodiment, the application includes preparing a medicament for the prevention, relief, and / or treatment of acute myocardial infarction and subsequent ventricular remodeling by mixing Trichinella spiralis cysteine ​​protease inhibitor Cystatin with pharmaceutical excipients.

[0017] In one embodiment, the pharmaceutical excipient comprises fillers, binders, wetting agents, disintegrants, lubricants, and / or flavoring agents.

[0018] In one embodiment, the filler is starch, sucrose, lactose, calcium sulfate, and / or microcrystalline cellulose; the binder is a cellulose derivative, alginate, gelatin, and / or polyvinylpyrrolidone; the wetting agent is water, ethanol, starch, and / or syrup; the disintegrant is sodium carboxymethyl starch, carboxypropyl cellulose, croscarmellose, agar, calcium carbonate, and / or sodium bicarbonate; the lubricant is talc, calcium stearate, magnesium stearate, microcrystalline silica gel, and / or polyethylene glycol; and the flavoring agent is simple syrup, sucrose, lecithin, orange peel syrup, cherry syrup, lemon, fennel, peppermint oil, sodium alginate, gum arabic, gelatin, methylcellulose, sodium carboxymethyl cellulose, citric acid, tartaric acid, and / or sodium bicarbonate.

[0019] In one embodiment, the drug further comprises a pharmaceutically acceptable drug carrier.

[0020] In one embodiment, the drug carrier comprises microcapsules, microspheres, nanoparticles, and / or liposomes.

[0021] In one embodiment, the route of administration of the drug includes intradermal injection, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intravenous infusion, arterial injection, intracavitary injection, and / or oral administration.

[0022] In one embodiment, the dosage form of the drug is powder, granules, capsules, tablets, pills, or oral liquid.

[0023] In one embodiment, the drug may also include other active ingredients with similar pharmacological activities.

[0024] Beneficial effects:

[0025] Expanding the therapeutic field of "worm therapy" (cardiovascular diseases) and further refining the molecular mechanism of worm immunomodulatory effects, this invention demonstrates the preventive and therapeutic effects of rTs-Cys on mice with acute myocardial infarction. This provides a new strategy for the prevention and treatment of other common metabolic or immune-related diseases. The invention confirms that rTs-Cys reduces mortality and cardiac index in acute myocardial infarction; reduces infarct size; decreases the degree of myocardial fibrosis after myocardial infarction and delays cardiac function impairment; inhibits pro-inflammatory cytokine levels and increases the expression of immunomodulatory factors; reduces inflammatory cell infiltration in the infarct junction of MI mice; and reduces cardiac troponin T levels in mice with acute myocardial infarction. Based on the therapeutic mechanism and improvement of various clinical indicators of worm therapy in multiple disease models, this invention aims to optimize cardiovascular disease treatment plans to achieve better therapeutic effects, restart vitality, and enjoy a new life. Meanwhile, for other common metabolic or immune-related diseases, timely intervention with novel immunomodulatory drugs can greatly reduce the economic burden on patients, improve their quality of life, and promote population health. Furthermore, the solution of this invention can realize the "repurposing of old drugs," which can greatly shorten the time from drug discovery to clinical translation. Attached Figure Description

[0026] Figure 1 Electrophoresis image of purified rTs-Cys;

[0027] Figure 2 Changes in survival rate of mice in each group 28 days after modeling (n=5 / group, **P<0.01 vs MI+PBS group);

[0028] Figure 3 Changes in cardiac function in mice at 7, 14, and 28 days after modeling; A: Typical echocardiography (long-axis B-mode and pulsed Doppler); BG: Systolic function indices; H: Diastolic function indices; I: Heart rate changes in mice at the time of image acquisition; (n = 5 / group, *P < 0.05, **P < 0.01, ***P < 0.001 vs MI+PBS group);

[0029] Figure 4 Changes in heart-to-body weight ratio in mice at 7, 14, and 28 days after modeling (n = 5 / group, **P < 0.01, ***P < 0.001 vs MI+PBS group);

[0030] Figure 5 Changes in cardiac morphology in mice at 7, 14, and 28 days after modeling;

[0031] Figure 6 Changes in cardiac structure and fibrosis in mice at 7, 14, and 28 days after modeling; A: Representative images of coronary sections of the heart stained with Masson staining in each group of mice (15×, Scalebar: 1000μm; 200×, Scalebar: 1000μm); BD: Changes in left ventricular infarct wall thickness, percentage of left ventricular infarct area, and percentage of fibrosis area in mice after MI surgery (n = 4-6 / group, **P<0.01, ***P<0.001 vs MI+PBS group);

[0032] Figure 7 Typical HE staining results of the myocardial infarction junction area in mice at 7, 14 and 28 days after modeling (200×, Scalebar: 100μm);

[0033] Figure 8 The changes in cardiac troponin T were detected by immunofluorescence at 7, 14 and 28 days after modeling in mice of each group; A: Representative immunofluorescence images of cardiac troponin T in coronary sections of the heart of mice of each group (40×, Scalebar: 1000μm) B: Cardiac troponin T levels in the infarct area of ​​the heart of mice of each group (n=4, *P<0.05, **P<0.01, ***P<0.001 vs MI+PBS group);

[0034] Figure 9 Changes in pro-inflammatory factors and immunomodulatory factors in mice at 7, 14, and 28 days after modeling (n = 5 / group, ***P < 0.001 vs MI+PBS group); Detailed Implementation

[0035] Preparation and purification of rTs-Cys (Nanjing Zhongding Biotechnology Co., Ltd.):

[0036] A PAS (PCR-based Accurate Synthesis) method was used to design full-length splicing primers, with a protective base synthesis gene BQ692489.1 designed at each end of the primers. These primers were ligated into the expression vector pCzn1 via cloning sites Ndel and Xbal. The resulting recombinant plasmid pCzn1-BQ692489.1 was transformed into TOP10 cloning strains and prokaryotic host Arctic-Express (DE3) competent cells. Expression of the target protein was induced at 15°C using 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The recombinant protein BQ692489.1 (rTs-Cys) in the supernatant at 15°C was purified using Ni column affinity purification. SDS-PAGE electrophoresis showed that its size was approximately 45 kDa. Figure 1The prepared rTs-Cys was stored at -80℃ for later use.

[0037] Example 1. Effect of rTs-Cys on the survival rate of mice with acute myocardial infarction

[0038] (1) Experimental materials:

[0039] Animals: Male C57BL / 6J mice, 8-10 weeks old, SPF grade, weighing approximately 20-25g, purchased from the Experimental Animal Center of Bengbu Medical College. Mice were housed in a standard animal facility with free access to food and water, and under controlled lighting conditions. The animal experimental procedures were approved by the ethics committee and complied with animal ethics standards (LAEC-2014-039).

[0040] Instrument: Reward small animal inhalation anesthesia machine.

[0041] Reagent: Isoflurane.

[0042] (2) Experimental methods:

[0043] The "Gao et al. Circ Res, 2010" model was used: After anesthesia and disinfection with the gaseous anesthetic isoflurane, the mice were fixed in a supine position. An oblique incision was made in the 3rd-4th intercostal space on the left anterior chest wall. The anterior chest wall muscle tissue was bluntly dissected. The thorax was compressed with the left hand, and the heart was gently compressed in the 4th intercostal space with hemostatic forceps in the right hand and placed outside the thoracic cavity. The left anterior descending coronary artery was ligated (location: the point where the upper margin of the left atrium and the main venous trunk are equidistant). After the apex of the heart turned grayish-white, the heart was quickly repositioned, the thoracic cavity was compressed to expel air, and the incision was sutured closed. Electrocardiogram showing signs of myocardial ischemia confirmed the successful modeling. After modeling, the acute myocardial infarction period (MI) in mice was from day 0 to 7, the fibrosis period was from day 8 to 14, and the ventricular remodeling period (VR) was from day 15 to 28.

[0044] Day 0 was defined as the day of model establishment. The experiment was divided into 4 groups, with 6 mice in each group: ① Negative control group (Sham+PBS group): no model establishment was performed, and PBS was injected intraperitoneally on days 1, 3, and 5; ② rTs-Cys control group (Sham+rTs-Cys): no model establishment was performed, and rTs-Cys 25 μg / mouse was injected intraperitoneally on days 1, 3, and 5; ③ MI and VR model group (MI+PBS): PBS was injected intraperitoneally on days 1, 3, and 5 after model establishment; ④ MI and VR treatment group (MI+rTs-Cys): rTs-Cys 25 μg / mouse was injected intraperitoneally on days 1, 3, and 5 after model establishment.

[0045] Observe and record the survival rate of mice in each group 28 days after modeling.

[0046] (3) Experimental results:

[0047] The survival status of mice in each group was observed for 28 days. It was found that the majority of deaths occurred within 7 days after MI surgery. The results showed no significant difference in survival rate between the Sham+PBS group and the Sham+rTs-Cys group. Within 28 days after MI surgery, the survival rate of mice in the MI+PBS group was 70%, significantly lower than that of the Sham+PBS group (100%); the survival rate of mice in the MI+rTs-Cys group was 100%, significantly higher than that of the MI+PBS group. These results indicate that rTs-Cys protein can significantly improve the survival rate of mice after MI surgery. Figure 2 Furthermore, rTs-Cys is safe and non-toxic to mice.

[0048] Example 2. Effects of rTs-Cys on cardiac function in mice with acute myocardial infarction

[0049] (1) Experimental materials:

[0050] Animals: Same as in Example 1.

[0051] Equipment: Reward small animal inhalation anesthesia machine, small animal ultrasound imaging system (VisualSonics, Canada).

[0052] Reagent: Isoflurane.

[0053] (2) Experimental method: Ultrasound was performed on mice 7, 14 and 28 days after modeling. Mice were anesthetized with isoflurane and fixed on the ultrasound operating table. Hair was removed from the left anterior chest with hair removal cream and ultrasound coupling agent was applied. Echocardiography was performed on mice under stable anesthesia. Left ventricular systolic function was assessed by analyzing stroke volume (SV), left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), cardiac output (CO), left ventricular end-systolic volume (LVESV), and left ventricular end-diastolic volume (LVEDV) using the LV trace method in long-axis B-mode. Pulsed Doppler recordings were performed in four-chamber mode to analyze the peak early diastolic filling (E-peak) and peak end-diastolic filling (A-peak) of the mitral valve, and the E / A ratio was calculated to reflect left ventricular diastolic function.

[0054] (3) Experimental results:

[0055] Compared with the Sham+PBS group, the heart morphology of the Sham+rTs-Cys group showed no significant changes; the heart chambers of the MI+PBS group were significantly enlarged and rounded, with a pear-shaped or spherical shape, and the mobility of the anterior and posterior walls of the left ventricle was significantly reduced; compared with the MI+PBS group, the heart chambers of the MI+rTs-Cys group were smaller, and the morphology of the heart chambers and the wall motion of the ventricle were significantly improved. Figure 3 A).

[0056] Statistical results showed that compared with the Sham+PBS group, there was no significant difference in cardiac function in the Sham+rTs-Cys group. In the MI+PBS group, at 7, 14, and 28 days after MI surgery, SV, CO, LVEF, and LVFS were significantly decreased, while LVESV and LVEDV were significantly increased, indicating a significant decrease in left ventricular systolic function. Compared with the MI+PBS group, in the MI+rTs-Cys group, at 7, 14, and 28 days after MI surgery, SV, CO, LVEF, and LVFS were significantly increased, while LVESV and LVEDV were significantly decreased, indicating a significant improvement in left ventricular systolic function. Figure 3 BG). Compared with the Sham+PBS group, the E / A ratio of mice in the MI+PBS group was significantly decreased at 7, 14, and 28 days after MI surgery; the E / A ratio of mice in the MI+rTs-Cys group showed an increasing trend at 7 and 14 days after MI surgery, with no statistically significant difference, but the E / A ratio was significantly increased at 28 days after MI surgery, indicating a significant improvement in left ventricular diastolic function. Figure 3 H). During echocardiography, the heart rate of the mice was maintained between 460-480 BPM, with no significant difference between groups, ensuring that the cardiac function of the mice was not affected by changes in heart rate. Figure 3 I). The above results indicate that rTs-Cys can significantly improve cardiac dysfunction caused by acute myocardial infarction and subsequent excessive ventricular remodeling in MI mice.

[0057] Table 1. Cardiac function of mice in each group (7 days)

[0058]

[0059] Table 2. Cardiac function of mice in each group (14 days)

[0060]

[0061] Table 3. Cardiac function of mice in each group (28 days)

[0062]

[0063] Example 3. Effect of rTs-Cys on the heart-to-body weight ratio in mice with acute myocardial infarction

[0064] (1) Experimental materials:

[0065] Sample: Mouse hearts collected after the mice in Example 2 were euthanized.

[0066] Instrument: Micro electronic scale

[0067] Reagent: PBS

[0068] (2) Experimental methods:

[0069] After the ultrasound imaging measurement in Example 2, the weight of mice in each group was weighed and recorded. Blood was collected from the eyeballs, the thoracic cavity of the mice was opened, and the heart was perfused with PBS through the aorta to remove blood. The heart was removed (excess tissue was removed, and the left and right atrial appendages were preserved), washed again with PBS, dried with filter paper, and the weight of the heart was measured with a micro-scale electronic scale to calculate the heart-to-body weight ratio.

[0070] (3) Experimental results:

[0071] Compared with the Sham+PBS group, rTs-Cys did not cause a significant change in the heart-to-body weight ratio in mice, while the heart-to-body weight ratio in the MI+PBS group increased significantly after surgery; after rTs-Cys treatment, the heart-to-body weight ratio in mice did not change significantly at 7 days post-surgery, but decreased significantly at 14 and 28 days post-surgery, which was statistically significant. Figure 4 The heart-to-body weight ratio is calculated as follows: Heart-to-body weight ratio = Heart weight (mg) / Body weight (g)

[0072] The above results indicate that rTs-Cys can reduce the heart-to-body weight ratio in mice with acute myocardial infarction.

[0073] Table 4. Heart-to-body weight ratio of mice in each group

[0074]

[0075] Example 4. Effects of rTs-Cys on cardiac morphology in mice with myocardial infarction

[0076] (1) Experimental materials:

[0077] Sample: Mouse heart tissue collected in Example 3

[0078] Instrument: Stereo microscope.

[0079] (2) Experimental methods:

[0080] The morphology of the heart was observed and recorded under a microscope.

[0081] (3) Experimental results:

[0082] Compared with the Sham+PBS group, rTs-Cys did not cause significant changes in the morphology of the mouse heart. However, after MI modeling, the mouse heart enlarged, the infarct area was clearly visible, and the ventricular wall of the infarct area gradually thinned over time. Compared with the MI+PBS group, the heart of the mouse in the MI+rTs-Cys group was smaller, and the infarct area was reduced. This indicates that rTs-Cys has a positive effect on the cardiac morphology of mice with acute myocardial infarction. Figure 5 ).

[0083] Example 5. Effects of rTs-Cys on cardiac structure and myocardial fibrosis after myocardial infarction in mice.

[0084] (1) Experimental materials:

[0085] Sample: Mouse heart tissue collected in Example 3

[0086] Instrument: Microscope (Nikon, Japan)

[0087] Reagents: Xylene, anhydrous ethanol, 75% alcohol, glacial acetic acid, paraffin, Masson staining kit (Servicebio, China)

[0088] (2) Experimental methods:

[0089] The collected cardiac tissue was washed with pre-cooled PBS and then fixed by immersion in 4% paraformaldehyde. After paraffin embedding and sectioning, the tissue was dewaxed and hydrated, and stained using the Masson staining method. The slides were read and images were acquired under a microscope. The left ventricular wall thickness, the percentage of left ventricular infarct size, and the percentage of fibrosis area were measured using ImageJ software.

[0090] (3) Experimental results:

[0091] As time progressed after MI surgery, the thickness of the left ventricular infarct wall in mice showed a decreasing trend, while the infarct area and fibrosis area of ​​the left ventricle showed an increasing trend. Figure 6 A). Specifically, compared with the MI+PBS group, mice in the MI+rTs-Cys group showed significant thickening of the left ventricular infarct area wall at 7, 14, and 28 days post-surgery; a significantly reduced left ventricular infarct area; and no significant change in the left ventricular fibrosis area at day 7, but significant improvement at days 14 and 28. Figure 6 There was no significant difference between the Sham+PBS group and the Sham+rTs-Cys group (BD).

[0092] The above results indicate that rTs-Cys can protect ventricular compliance and ventricular remodeling after MI by delaying the thinning of the ventricular wall in the left ventricular infarct area, reducing the left ventricular infarct area, and decreasing the degree of left ventricular fibrosis.

[0093] Table 5. Mouse heart structure and degree of myocardial fibrosis (7 days)

[0094]

[0095] Table 6. Mouse heart structure and degree of myocardial fibrosis (14 days)

[0096]

[0097] Table 7. Mouse heart structure and degree of myocardial fibrosis (28 days)

[0098]

[0099] Example 6. Effects of rTs-Cys on myocardial injury and inflammatory cell infiltration in the infarct junction of MI mice.

[0100] (1) Experimental materials:

[0101] Sample: Mouse heart tissue collected in Example 3

[0102] Instrument: Microscope (Nikon, Japan)

[0103] Materials: Oil Red O dye, hematoxylin & eosin dye (HE) (China Servicebio)

[0104] (2) Experimental methods:

[0105] The heart tissue collected in Example 3 was washed with pre-cooled PBS and fixed with 4% paraformaldehyde. After sectioning, the tissue was stained with hematoxylin and eosin and read under a microscope.

[0106] (3) Experimental results:

[0107] HE staining was used to observe the degree of myocardial injury and inflammatory cell infiltration in the infarct junction of MI mice. The corresponding locations were observed in the Sham+PBS group and the Sham+rTs-Cys group. Results showed that compared with the Sham+PBS group, there were no significant changes in the Sham+rTs-Cys group; in the MI+PBS group, inflammatory cells significantly accumulated and infiltrated in the infarct junction, cardiomyocytes vacuolated, and myocardial fiber ruptured. Compared with the MI+PBS group, the MI+rTs-Cys group showed reduced inflammatory cell infiltration, reduced cardiomyocyte vacuolation and tissue damage, improved tissue structure, and decreased degree of myocardial fiber rupture in the infarct junction. Figure 6Histopathological results showed that rTs-Cys could alleviate myocardial injury by reducing inflammatory cell infiltration at the myocardial infarction junction in MI mice.

[0108] Example 7. Effect of rTs-Cys on cardiac troponin T (CTNT) levels after myocardial injury in mice.

[0109] (1) Experimental materials:

[0110] Sample: Mouse heart tissue collected in Example 3

[0111] Materials: Primary antibody (Ablonic antibody, China), fluorescent secondary antibody (Ablonic antibody, China)

[0112] Instrument: Upright fluorescence microscope

[0113] (2) Experimental methods:

[0114] The collected heart tissue was washed with pre-cooled PBS and then fixed by soaking in 4% paraformaldehyde. After being embedded in paraffin and sectioned, the tissue was dewaxed to water and then subjected to antigen retrieval. The autofluorescence was quenched by drawing circles, blocked with serum, and incubated overnight with primary antibody. After washing, secondary antibody was added and incubation continued. The cell nuclei were counterstained with DAPI, and the tissue was mounted and observed under a fluorescence microscope. Images were collected and analyzed.

[0115] (3) Experimental results:

[0116] Compared with the Sham+PBS group, there was no significant difference in the Sham+rTs-Cys group; the cardiac troponin T level in the MI+PBS group was significantly increased at 7, 14, and 28 days post-surgery. Compared with the MI+PBS group, the cardiac troponin T level in the MI+rTs-Cys group was significantly decreased at 7 and 14 days post-surgery, which was statistically significant; a decreasing trend was observed at 28 days post-surgery, but no significant statistical difference was observed. Figure 8 The above results indicate that rTs-Cys can reduce the level of cardiac troponin T in mice with acute myocardial infarction and alleviate myocardial damage.

[0117] Table 8. Cardiac troponin T levels in mice of each group

[0118]

[0119]

[0120] Example 8. Effects of rTs-Cys on the levels of pro-inflammatory and immunomodulatory cytokines in mice.

[0121] (1) Experimental materials:

[0122] Samples: Mouse serum and heart tissue collected in Example 3.

[0123] Materials: Tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) ELISA kit (Shanghai Dakewei);

[0124] Reverse transcription kit (TransGen Biotech, China), Real-time fluorescence kit (TransGen Biotech, China)

[0125] Instrument: Roche 96 real-time PCR system (USA).

[0126] (2) Experimental methods:

[0127] After collecting blood from the mouse eyeballs, the serum was collected by centrifugation at 4500 rpm for 15 min at 4℃ and stored at -80℃ for subsequent ELISA detection.

[0128] Mouse heart tissue was rinsed with pre-cooled PBS, and an appropriate amount of tissue was weighed. Trizol lysis buffer was added, and the tissue was homogenized at 65 Hz for 60 seconds. Total RNA was extracted from the heart tissue following the prescribed steps. cDNA was then synthesized from 2 μg of total RNA using a reverse transcription kit. Using a quantitative real-time assay kit, 2 μl cDNA was used as a template, and the total sample volume was 20 μl. GAPDH was used as an internal control. The above analysis. Through 2 -△△Cq The relative mRNA expression levels of the above-mentioned cytokines in cardiac tissue were detected.

[0129] (3) Experimental results:

[0130] The ELISA kit was used to detect the serum secretion levels of inflammatory factors (TNF-α, IL-10) in mice of each group, and the PT-PCR technique was used to detect the mRNA expression levels of corresponding inflammatory cytokines in the myocardial infarction area of ​​mice of each group at different time points (the apical area was detected in the Sham+PBS group and the Sham+rTs-Cys group).

[0131] The results showed that compared with the Sham+PBS group, the MI+PBS group mice had increased serum pro-inflammatory cytokine TNF-α levels within 28 days after MI surgery, reaching a peak at day 14. Compared with the MI+PBS group, the MI+rTs-Cys group mice showed significantly decreased serum pro-inflammatory TNF-α levels and significantly upregulated immunomodulatory cytokine IL-10 levels at days 7, 14, and 28. The mRNA expression levels of pro-inflammatory TNF-α and immunomodulatory cytokine IL-10 in cardiac tissue were consistent with those in serum. These results indicate that rTs-Cys treatment can significantly inhibit the expression of pro-inflammatory factors and promote the expression of immunomodulatory cytokines, thereby protecting myocardial tissue from inflammatory attacks.

[0132] Table 9. Serum secretion levels of inflammatory factors (TNF-α, IL-10) in mice (7 days)

[0133]

[0134]

[0135] Table 10. Serum secretion levels of inflammatory factors (TNF-α, IL-10) in mice (14 days)

[0136]

[0137] Table 11. Serum secretion levels of inflammatory factors (TNF-α, IL-10) in mice (28 days)

[0138]

[0139] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. The use of Cystatin, a Trichinella spiralis cysteine ​​protease inhibitor, in the preparation of drugs for relieving and / or treating acute myocardial infarction and for relieving ventricular remodeling after acute myocardial infarction, characterized in that... The nucleotide sequence of Cystatin is shown in SEQ ID NO.1, and the relief and / or treatment of acute myocardial infarction and the relief of ventricular remodeling after acute myocardial infarction include at least one of the following: (1) Reduce the mortality rate and cardiac index of acute myocardial infarction; (2) Reduce the heart-to-body weight ratio and the area of ​​myocardial infarction; (3) Reduce the degree of myocardial fibrosis after myocardial infarction and delay cardiac function impairment; (4) Inhibit the levels of pro-inflammatory cytokines and increase the expression of immune regulatory factors; (5) Reduces inflammatory cell infiltration at the myocardial infarction junction in MI mice; (6) Reduce the level of cardiac troponin T in mice with acute myocardial infarction.

2. The application according to claim 1, characterized in that, Cystatin is used alone as the active ingredient in the drug.

3. The application according to claim 1, characterized in that, The applications include preparing drugs for relieving and / or treating acute myocardial infarction and relieving ventricular remodeling after acute myocardial infarction by mixing Trichinella spiralis cysteine ​​protease inhibitor Cystatin with pharmaceutical excipients.

4. The application according to claim 3, characterized in that, The pharmaceutical excipients include fillers, binders, wetting agents, disintegrants, lubricants, and / or flavoring agents.

5. The application according to claim 4, characterized in that, The filler is starch, sucrose, lactose, calcium sulfate, and / or microcrystalline cellulose; the binder is cellulose derivative, alginate, gelatin, and / or polyvinylpyrrolidone; the wetting agent is water, ethanol, starch, and / or syrup; the disintegrant is sodium carboxymethyl starch, carboxypropyl cellulose, croscarmellose, agar, calcium carbonate, and / or sodium bicarbonate; the lubricant is talc, calcium stearate, magnesium stearate, micronized silica gel, and / or polyethylene glycol; the flavoring agent is simple syrup, sucrose, lecithin, orange peel syrup, cherry syrup, lemon, fennel, peppermint oil, sodium alginate, gum arabic, gelatin, methylcellulose, sodium carboxymethyl cellulose, citric acid, tartaric acid, and / or sodium bicarbonate.

6. The application according to claim 1, characterized in that, The drug also contains a pharmaceutically acceptable drug carrier.

7. The application according to claim 6, characterized in that, The drug carrier comprises microcapsules, microspheres, nanoparticles, and / or liposomes.

8. The application according to any one of claims 1 to 7, characterized in that, The dosage form of the drug is powder, granules, capsules, tablets, pills, or oral liquid.

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