CN113559245A

CN113559245A - Application of teduglutide in preparation of medicine for treating myocardial ischemia reperfusion injury

Info

Publication number: CN113559245A
Application number: CN202111008332.0A
Authority: CN
Inventors: 谢峻; 赵金璇; 程伟
Original assignee: Nanjing Drum Tower Hospital
Current assignee: Nanjing Drum Tower Hospital
Priority date: 2021-08-31
Filing date: 2021-08-31
Publication date: 2021-10-29

Abstract

The invention discloses an application of teduotide in preparing a medicine for treating myocardial ischemia-reperfusion injury, wherein the teduotide can protect the integrity of an intestinal barrier after myocardial ischemia, prevent intestinal flora shift and intestinal flora imbalance and relieve myocardial inflammatory reaction after myocardial ischemia-reperfusion so as to treat myocardial ischemia-reperfusion injury. The invention discovers that the teduglutide has the effect of treating myocardial ischemia reperfusion injury after myocardial infarction, can be used for preparing a medicament for treating acute myocardial infarction, and provides a new treatment scheme for clinically treating the myocardial ischemia reperfusion injury.

Description

Application of teduglutide in preparation of medicine for treating myocardial ischemia reperfusion injury

Technical Field

The invention belongs to the field of medicines, and particularly relates to an application of teduotide in preparation of a medicine for treating myocardial ischemia-reperfusion injury.

Background

With the economic development and the improvement of the living standard of people, the number of patients suffering from Acute Myocardial Infarction (AMI) is increased year by year, and the disease becomes the leading cause of death and disability in China. Acute myocardial infarction is a myocardial necrotizing disease caused by acute occlusion of coronary arteries, and rapid and complete opening of occluded blood vessels is a main treatment means of the acute myocardial infarction. Since the 21 st century, with the popularization of emergency coronary intervention, more and more AMI patients get timely revascularization treatment, and the death rate of AMI is obviously reduced. However, reperfusion injury causes further death of a large number of myocardial cells, so that severe cardiac hypofunction appears in the late stage of myocardial infarction patients, and the long-term prognosis is influenced. There is a large body of evidence that an overactivated immune inflammatory response after myocardial ischemia reperfusion plays an important role in its development. After myocardial ischemia reperfusion, a moderate inflammatory response is beneficial to clearing necrotic cells and extracellular matrix in the infarct area, and clearing the disorder for the subsequent proliferation and repair. Further myocardial cell damage may result if the myocardial inflammatory response is too severe or persists.

In recent years the role of human intestinal microorganisms in cardiovascular diseases and metabolic disorders has attracted increasing attention. Subsequent studies have indicated that there is a structural disturbance and an imbalance in the proportion of the intestinal flora after myocardial infarction. A Chua Jun professor team sends a text to report that the phenomenon of intestinal flora shift and circulating Lipopolysaccharide (LPS) rise exists after acute myocardial infarction for the first time, and the amplification of the LPS in the circulation of an acute ST-segment elevation myocardial infarction patient is positively correlated with the amplification of proinflammatory monocytes in the circulation. Studies by foreign scholars further demonstrate that the level of LPS in circulation in acute ST-elevation myocardial infarction patients is independently correlated with the level of zonulin, a marker of intestinal permeability in circulation. The applicant also finds that the damage of the intestinal form, permeability and barrier function, the migration of intestinal flora to the circulation and the increase of LPS in the circulation exist after the myocardial ischemia reperfusion of mice, and the change trend of the intestinal flora ectopy is the same as the change trend of proinflammatory macrophages in the damaged myocardium, and the peak is reached on the third day after the operation. In contrast, in the case of the aseptic mice lacking intestinal flora, the tissue damage after myocardial ischemia reperfusion is significantly reduced compared with that in the wild mice. These results suggest that the shift of intestinal flora is closely related to the activation and persistence of inflammatory reaction after myocardial ischemia, and have important significance on inflammatory reaction after myocardial ischemia reperfusion. Therefore, the method for reducing the ectopic and the imbalance of intestinal flora after myocardial ischemia reperfusion is found, and the method plays an important role in clinically relieving myocardial ischemia reperfusion injury.

Antibiotic therapy theoretically could eliminate the intestinal flora to cut the link between intestinal flora and cardiovascular disease. However, the application of broad-spectrum antibiotics increases the risk of heart rupture after myocardial infarction, and the combination of broad-spectrum antibiotic treatment increases the risk of drug-resistant bacteria and fungal infection, so that the 'simple and rough' antibiotic therapy is difficult to apply clinically.

The dietary intervention or probiotic therapy can play a role in protecting intestinal immune barriers by changing intestinal microecology, and has good application prospect in the treatment of chronic diseases such as diabetes, obesity, atherosclerosis and the like. It is clear that this long-term treatment pattern and the need for advanced intervention greatly limits its use in patients with acute myocardial infarction. In recent years, fecal transplantation has attracted more and more attention as a new treatment for improving intestinal flora disturbance and protecting intestinal barrier, and has been widely used for treating intestinal diseases and partial parenteral diseases. However, because of its complex composition, it has the potential risk of causing the transfer of endotoxin or infectious agents and inducing new gastrointestinal complications, and the therapeutic effect and safety are difficult to be effectively ensured. Therefore, there is an urgent need to find new safe and effective therapeutic means for preventing the shift of intestinal flora after myocardial ischemia-reperfusion.

Glucagon-like Peptide 2 (GLP-2), a polypeptide consisting of 33 amino acids, is secreted by L cells of the terminal ileum and colon and was originally discovered as an enteral trophic factor that promotes proliferation of the intestinal mucosa. In recent years, breakthrough researches show that the function of GLP-2 is not only limited to the intestinal tract, but also proved to be capable of relieving inflammatory injuries of the liver, the nervous system and the like, and the specific mechanism of the GLP-2 needs to be deeply researched. GLP-2 has extremely short half-life in vivo and is extremely easily degraded by dipeptidyl peptidase IV (DPP 4) in vivo. Therefore, a GLP-2 analogue which is called Teduglutide (Teduglutide) and is resistant to degradation by DPP4 enzyme is developed, the Teduglutide is formed by replacing the second position 'A (alanine-alanine)' with 'G (glycine-glycine)' on the basis of a GLP-2 sequence, and the generated (Gly2) -GLP-2 has the advantages of both efficacy and degradation resistance and obviously prolonged half life compared with the natural GLP-2. Teduglutide is currently approved by the U.S. FDA for marketing in 2012 for the treatment of intestinal failure.

Animal experiments prove that the teduglutide can protect the integrity of intestinal barriers after myocardial ischemia, prevent intestinal flora shift and intestinal flora imbalance, inhibit infiltration of neutrophilic granulocytes and proinflammatory monocytes in myocardial tissues and relieve myocardial inflammatory reaction after myocardial ischemia reperfusion for the first time. The invention provides a new idea for preventing and treating excessive activation of inflammatory response after myocardial ischemia reperfusion, and has higher clinical transformation value and clinical application realizability.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention discloses a new application of teduotide, and particularly discloses an application of teduotide in preparing a medicament for treating myocardial ischemia reperfusion after acute myocardial infarction vessel recanalization.

The invention provides an application of teduglutide in preparing a medicine for treating myocardial ischemia-reperfusion injury, which comprises the following specific steps: the application in preparing the medicine for treating the shift of intestinal flora and the inflammation storm caused by the imbalance of the intestinal flora caused by myocardial ischemia reperfusion after the recanalization of the acute myocardial infarction vessel;

wherein the myocardial ischemia reperfusion injury is further myocardial injury caused by revascularization treatment after acute coronary artery occlusion, and comprises oxidative stress injury, inflammatory response injury and the like;

wherein the inflammatory storm is the disturbance of intestinal flora damage caused by intestinal permeability and barrier function after myocardial ischemia reperfusion, the migration of intestinal flora to circulation and the penetration of LPS into blood are triggered, the activation and mobilization of inflammatory cells such as neutrophil granulocytes and mononuclear macrophages are promoted, a large amount of inflammatory factors and chemotactic factors are generated and secreted, the infiltration of proinflammatory cells to damaged myocardium is promoted, and the activation and continuous existence of inflammatory reaction after myocardial ischemia reperfusion are caused;

wherein, the teduglutide can be in the forms of raw material medicine, solvate and salt thereof, and other glucagon-like peptide-2 analogues with similar amino acid sequences, such as apraglutide;

wherein the amino acid sequence of apraglutide is as follows:

His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-norleucine-D-Phenylalanine-Thr-Ile-Leu-Asp-Leu-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-NH₂

the amino acid sequence of the teduglutide is as follows:

H-His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-OH。

in another aspect, the invention provides that the mice with teduglutide are administered twice a day at a dose of 600-;

according to a human and animal body surface area ratio dose table in pharmacological experiment methodology compiled by professor Xutu of Tertiary cloud, according to an equivalent dose ratio converted from the body surface area of a human and a mouse: the dose (mg/kg) administered to mice is equivalent to the dose (mg/kg) administered to humans x 70kg x 0.0026/0.02kg, so that the human dose of teduglutide is 66-132 μ g/kg, preferably 66 μ g/kg, twice daily.

Finally, the teduglutide can be prepared into a pharmaceutical preparation with a conventional pharmaceutical adjuvant in pharmaceutics;

wherein said pharmaceutical formulation can be administered parenterally;

wherein, the medicinal preparation is an injection preparation or a powder injection.

Has the advantages that:

the invention discovers that in a mouse myocardial ischemia reperfusion model constructed by restoring coronary blood flow after 60 minutes of debranching ischemia before ligating a mouse, 600 mu g/kg of teduglutide is subcutaneously injected twice a day after modeling for 3 consecutive days, the integrity of an intestinal barrier after myocardial ischemia can be protected, the intestinal flora shift and the intestinal flora imbalance are prevented, and the myocardial inflammatory reaction after myocardial ischemia reperfusion is relieved, so that the myocardial ischemia reperfusion injury is relieved, the cardiac function and the long-term ventricular remodeling are improved, the long-term survival rate of the mouse is improved, and the prognosis is improved. The invention discovers that the teduglutide has the functions of preventing the intestinal flora from shifting and the intestinal flora from being imbalanced after the myocardial ischemia reperfusion, relieving the myocardial inflammatory reaction after the myocardial ischemia reperfusion and protecting the cardiac function, can be used for preparing the treatment medicament for the myocardial ischemia reperfusion and provides a new treatment scheme for clinical treatment.

Drawings

FIG. 1 is a graph showing the effect of varying doses of teduglutide in example 1 on the treatment of myocardial ischemia reperfusion injury.

Fig. 2 is a graph showing that the teduglutide treatment in example 2 significantly improved cardiac function after myocardial ischemia-reperfusion.

Figure 3 is a graph showing that the survival rate of mice perfused with myocardial ischemia was significantly improved by the teduglutide treatment in example 2.

Figure 4 is a graph showing that the treatment with teduglutide in example 3 significantly reduced the formation of microcirculatory disturbance following myocardial ischemia-reperfusion.

FIG. 5 is a graph showing that the treatment with teduglutide in example 4 significantly reduced the levels of IL-6 and IL-1 β proinflammatory factors in damaged myocardium and in circulation following myocardial ischemia-reperfusion.

Fig. 6 is a graph showing that the treatment with teduglutide in example 5 significantly reduced neutrophil and macrophage infiltration in damaged myocardium after myocardial ischemia-reperfusion.

Fig. 7 is a graph showing that the treatment with teduglutide in example 6 significantly improved the disturbance of intestinal flora after myocardial ischemia-reperfusion.

Fig. 8 is a graph showing that the treatment with teduglutide in example 7 significantly inhibited the intestinal flora shift after reperfusion by myocardial ischemia.

Detailed Description

The present invention will be further described with reference to the accompanying drawings.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

The Teduglutide (Teduglutide) used in the following examples is all of the structure shown below with a purity of > 98%.

H-His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-OH

The teduglutide used in the examples of the present invention was purchased from Creative Peptide, ltd, cat #: 10-101-285. The animals used in the examples of the present invention were all C57BL/6 mice (SPF grade, 8 weeks old, male) purchased from the model animal institute at the university of tokyo.

EXAMPLE 1 Effect of varying doses of teduglutide on treatment of myocardial ischemia reperfusion injury

Materials: the small animal respirator, the animal operation illuminating lamp and the gas anesthesia system are purchased from Shanghai Yuyan scientific instruments, Inc.; the surgical micro-instrument is purchased from Zhen Mei medical instruments Co., Ltd; 4-0/7-0 suture was purchased from Ningbo medical needles Inc.; 2,3, 5-Triphenyltetrazolium chloride (TTC), Evans Blue (Evan & Blue) was purchased from Sigma, USA.

Constructing a mouse myocardial ischemia reperfusion model: male C57BL/6 mice at 8 weeks of age were randomly assigned to sham (sham), Saline (salt), teduglutide 300. mu.g/kg (300. mu.g/kg GLP-2), teduglutide 600. mu.g/kg (600. mu.g/kg GLP-2) and teduglutide 1200. mu.g/kg (1200. mu.g/kg GLP-2) groups, 5 each. The modeling method comprises the following steps: isoflurane anaesthetizes the mouse, connects breathing machine assisted respiration behind the trachea cannula, the preceding defeathering of chest, after iodophor epidermis is disinfected, cut in the third four intercostal department, expose the heart, use 7-0 line to untie the ligature line and resume the perfusion after 60min before ligature, sew up the incision with 4-0 suture, withdraw the breathing machine after the mouse spontaneous respiration resumes, the sham operation group only passes with the line, do not ligate, the different dose teducel group is injected the corresponding dose of isopyknal that resists DPP4 degradation into the subcutaneous injection immediately after reperfusion, twice a day, inject 3 days in succession, the isopyknal normal saline group is given at the same time.

Calculating the area of the myocardial stem by using Evan Blue and TTC dyeing: 3 days after myocardial ischemia reperfusion operation, mice are fully anesthetized by isoflurane, and limbs are fixed on an animal operation flat plate in a supine positionAnd (3) inserting a trachea, connecting a breathing machine, opening the chest along the original incision, separating layer by layer and exposing the heart. Ligating the coronary artery at the original ligation part of 1-2mm at the middle lower part of the left auricle with 7-0 suture again, separating and exposing the aorta of the mouse, killing the mouse by cervical dislocation, quickly cutting off the heart from the root of the aorta, placing the heart in a 10mL culture dish containing PBS, finding the opening of the aorta of the mouse, injecting PBS from the aorta by using a 1mL injector to lavage the heart, washing off residual blood in the heart, and observing the heart beating after the PBS is perfused. Then, a 1mL syringe was used to inject 5 units of 1% Evan Blue dye solution from the aorta, while the aortic orifice was clamped with a vascular clamp to prevent leakage of the dye solution, and after Evan Blue was injected, the region outside the visible descending branch blood supply was rapidly Blue-stained. The heart of a mouse is placed on a preservative film, the mouse is frozen for 15 minutes in a refrigerator at minus 80 ℃, the mouse heart is cut into 5-6 myocardial slices with the thickness of about 1mm by a blade and a mould according to the long axis direction, the myocardial slices are placed in a culture dish, the color floating is removed by flushing physiological saline, the myocardial slices are soaked in 1.5 percent TTC staining solution, and the mouse heart is incubated for 15 minutes at room temperature in a dark place. TTC can react with succinate dehydrogenase in mitochondria of viable cardiomyocytes to generate brick-red janus, while dead cardiomyocytes cannot react with TTC and are pale. The myocardial slices were removed and placed in a clean petri dish containing a small amount of physiological saline. And observing the effect, taking a picture, and collecting images of the sections on two sides of the myocardium. The blue-stained areas are viable myocardium, the parts not covered by blue dye are ischemic myocardium, the brick red areas within the range are myocardium salvaged by reperfusion in the ischemic area, and the white areas are necrotic myocardium. Analyzing the myocardial infarction range by adopting Image Pro Plus software, manually tracing the total area of each myocardial (TA), the myocardial ischemia area (brick red + white area, area at risk, AAR) and the myocardial infarction area (Infarct area, IA) after opening an Image, averaging the data measured on the front side and the back side of the same myocardial, and respectively accumulating the TA, the AAR and the IA measured on all myocardial slices of the same mouse. To obtain T_TA，T_AAR，T_IAThe calculation formula is as follows: AAR% ═ T_AAR/T_TA；Infarct Size％＝T_IA/T_AAR。

As shown in figure 1, the myocardial infarction areas of the 600. mu.g/kg and 1200. mu.g/kg tedulitide groups are obviously smaller than those of the Saline group, and the myocardial infarction areas between the two dose groups have no significant difference, which indicates that the dose range of the tedulitide is 600-1200. mu.g/kg, and the administration dose of the tedulitide in the subsequent experiments is 600. mu.g/kg.

Example 2 the teduglutide treatment significantly improved cardiac function after myocardial ischemia reperfusion and increased survival rate.

Materials: the small animal respirator, the animal operation illuminating lamp and the gas anesthesia system are purchased from Shanghai Yuyan scientific instruments, Inc., the operation microscopic instrument is purchased from Zhen Mei medical instrument, Inc., the 4-0/7-0 suture line is purchased from Ningbo medical suture needle, Inc., and the small animal heart ultrasonic instrument is purchased from VisualSonics, Canada.

Heart function measurement with heart ultrasonic: male C57BL/6 mice, 8 weeks old, were randomly divided into sham (sham), Saline (salt), and teduglutide (600 μ g/kg GLP-2) groups, 9 mice each, and a model of myocardial ischemia reperfusion of the mice was constructed as described above, the sham group was threaded with a thread only without ligation, and the teduglutide group was injected subcutaneously with 600 μ g/kg of teduglutide resistant to DPP4 degradation immediately after reperfusion, twice daily for 3 consecutive days. The normal saline group is given with the same volume of normal saline at the same time, and the heart ultrasonic detection is carried out on the mice before operation and after 3 days and 3 weeks after myocardial ischemia reperfusion. After isoflurane anesthesia, a mouse is fixed on a fixed plate with electrocardiographic detection, after Left chest hair is removed slightly, a Canada VisualSonics Vevo 2100 model small animal high-frequency color ultrasonic instrument is used, a Left Ventricular (LV) long axis section and a short axis section of the mouse are taken, M ultrasound is taken for measurement under the guidance of two-dimensional images, each section is selected to be measured repeatedly three times by selecting different cardiac cycles, and the inner diameter (LV end-systole diameter, LVID; d) of the Left ventricular end-diastole, the inner diameter (LV end-diastole diameter, LVID; s) of the Left ventricular end-systole, the thickness (IVS) of the ventricular interval of the end-systole and the thickness (LVP wall thickness, LVPW) of the Left ventricular end-diastole are measured respectively. Left ventricular short axis shortening (FS%) (LVID; d-LVID; s)/LVID; d × 100 (%); left ventricular Ejection fraction (Ejection fraction)n，EF％)＝(LV Vol；d-LV Vol；s)/LV Vol；d×100(％)；LV Vol；d＝((7.0/(2.4+LVID；d))×LVID；d³)；LV Vol；s＝((7.0/(2.4+LVID；s))×LVID；s³)。

And (3) survival rate statistics: male C57BL/6 mice, 8 weeks old, were randomly divided into sham (sham), Saline (salt), and teduglutide (600 μ g/kg GLP-2) groups, 10 mice each, and a mouse myocardial ischemia-reperfusion model was constructed as described above, and the sham group was threaded with a thread only without ligation. The teduglutide group was injected subcutaneously with 600 μ g/kg of teduglutide resistant to degradation by DPP4, twice daily for 3 consecutive days immediately after reperfusion. The saline group was given an equal volume of saline at the same time. And observing the survival rate of each group of mice under the same survival environment, and drawing a survival curve.

As shown in fig. 2, the teduglutide can significantly increase the left ventricular Ejection fraction (EF%) and the left ventricular short axis shortening (FS%) after myocardial ischemia reperfusion, and improve cardiac function. As shown in fig. 3, the teduglutide group had a higher survival rate than the saline group.

Example 3 treatment with teduglutide significantly reduced the formation of microcirculatory disturbance (MVO) following myocardial ischemia reperfusion

Materials: small animal ventilators, animal surgery lights, and gas anesthesia systems were purchased from Shanghai Yuyan scientific instruments, Inc. The surgical micro-instrument is purchased from Zhen Mei medical instruments Co. 4-0/7-0 suture was purchased from Ningbo medical needles, Inc. Thioflavin S dye was purchased from Biosharp corporation.

MVO area was calculated using thioflavin S staining: male C57BL/6 mice, 8 weeks old, were randomly assigned to sham (sham), Saline (Saline), and teduglutide (600. mu.g/kg GLP-2) groups, 5 each. Mouse myocardial ischemia reperfusion model was constructed as described above, and sham groups were threaded with only thread and not ligated. The teduglutide group was injected subcutaneously with 600 μ g/kg of teduglutide resistant to degradation by DPP4 immediately after reperfusion, twice daily for 3 consecutive days, and the saline group was given an equal volume of saline at the same time. Myocardial ischemia3 days after reperfusion, mice were fully anesthetized with isoflurane and then placed in supine position to fix the limbs on the animal surgery plate. The trachea cannula is connected with a breathing machine. The chest is opened along the original incision, the layer is separated layer by layer, the aorta of the mouse is exposed by separation, the mouse is killed by cervical dislocation, the heart is cut and taken out from the root part of the aorta rapidly, the heart is placed in a 10mL culture dish containing PBS, the opening of the aorta of the mouse is found, PBS is injected from the aorta by a 1mL injector to lavage the heart, residual blood in the heart is washed away, the heart beats can be seen after the PBS is infused, then 4 percent of thioflavin S staining solution is injected from the aorta by 5 units by the 1mL injector, the opening of the aorta is clamped by a vascular clamp during the process, the staining solution is prevented from leaking, the visible part of the heart is covered by faint yellow dye, the heart of the mouse is placed on a preservative film, the refrigerator is frozen for 15 minutes at minus 80 ℃, a blade and a mould are used for cutting into 5-6 myocardial slices with the thickness of about 1mm along the long axis direction, the effect is observed and photographed under an ultraviolet lamp, the section images of two sides of the myocardium are collected, the fluorescent blue area is the bonding area of the thioflavin S and the endothelial cell, here, the area is a blood recirculation area after reperfusion, and the black area is an area where blood flow is not recovered after reperfusion, namely a microcirculation disturbance (MVO) area, the myocardial infarction range is analyzed by using Image Pro Plus software, and the total area of the left ventricle of each myocardial (fluorescent blue left ventricle area, TLA) and the MVO area (black area, MVO area, MA) are manually traced after an Image is opened. Averaging the measured data of the front and back sides of the same myocardial, and respectively accumulating the TLA and MA measured by all myocardial slices of the same mouse to obtain T_TLA，T_MAThe calculation formula is as follows: MVO% ═ T_MA/T_TLA。

As shown in fig. 4, teduglutide significantly reduced the formation of microcirculatory disturbance following myocardial ischemia-reperfusion.

Example 4 treatment with teduglutide significantly reduced the levels of IL-6 and IL-1 β proinflammatory factors in damaged myocardium and in circulation following myocardial ischemia reperfusion

Materials: the small animal respirator, the animal operation illuminating lamp and the gas anesthesia system are purchased from Shanghai Yuyan scientific instruments, Inc., the operation microscopic instruments are purchased from Zhen Mei medical instruments, Inc., the 4-0/7-0 suture line is purchased from Ningbo medical suture needle, Inc., and the mice IL-6 and IL-1 beta ELISAKit are purchased from Union biotechnology, Inc.

And (3) blood and heart samples are taken: male C57BL/6 mice at 8 weeks of age were randomly divided into sham (sham), Saline (Saline), and teduglutide (600. mu.g/kg GLP-2) groups, 8 each. Mouse myocardial ischemia reperfusion model was constructed as described above, and sham groups were threaded with only thread and not ligated. The teduglutide group was injected subcutaneously with 600 μ g/kg of teduglutide resistant to degradation by DPP4, twice daily for 3 consecutive days immediately after reperfusion. The saline group was given an equal volume of saline at the same time. Mice were sacrificed 3 days post-surgery to leave heart and blood samples. After the mice are anesthetized with isoflurane, the eyeballs are picked up and blood is taken, the blood is reserved in a 1.5mL EP tube marked in advance, the mixture is kept still for 2 hours, then the centrifugation is carried out at 3000rpm for 5 minutes, and the upper layer serum is reserved and stored at minus 80 ℃ for subsequent experiments. After a mouse is removed from eyeballs and blood is taken, the mouse is killed by a cervical dislocation method, then the mouse is fixed on a mouse plate, KCl (2mol/L)1mL is injected into a heart cavity after chest opening is rapidly carried out, the heart of the mouse stops jumping in a diastole, a small opening is cut in a right atrium, physiological saline is injected from a left ventricle to carry out whole body perfusion, then the heart is cut off from the root part of an aorta carefully and taken out, the blood stain on the surface of the heart of the mouse is washed by the physiological saline, and gauze is wiped clean. The atrium and right ventricle portions were removed, the left ventricle tissue was quickly placed in a cryovial, placed in liquid nitrogen, and transferred to a-80 ℃ freezer for storage.

Enzyme-linked immunosorbent assay: using an ELISA kit to detect the content of IL-6 and IL-1 beta, taking out a detection sample from a refrigerator at a temperature of-80 ℃ before detection, thawing the detection sample on ice, weighing 50mg of tissues if the detection sample is a blood serum sample, adding 9 times of a homogenizing medium, homogenizing the tissue by using a tissue homogenizer (homogenizing time is 10 seconds/time, interval is 30 seconds, continuous 3-5 times and is carried out in ice water), centrifuging the grinding liquid for 10 minutes at 5000rpm, taking supernatant to prepare 10% of tissue homogenate, taking a small amount of homogenate to extract protein, measuring the protein concentration of each group of homogenate samples by using a BCA method, and facilitating subsequent statistical analysis of data, wherein the ELISA detection specific operation steps are as follows: (1) preparing a standard substance: the Standard lyophilized powder in the kit was added to a volume of "Standard/Sample Diluent" and gently shaken to dissolve it well. Sequentially diluting the standard substance according to the proportion in the specification, and preparing the standard substance with different concentration gradients; (2) 50 μ L of "ELISADiluent" was added to each well in 96-well plates. (3) Sample adding: adding 100 μ L of Standard with different concentrations into the Standard hole, adding 2 times diluted Sample (serum Sample can be undiluted) into the Sample hole, sticking a sealing plate membrane, and incubating in a constant temperature oven at 37 deg.C for 2 hr; (4) preparing a Wash Buffer: 100mL of 20 XWash Buffer +1900mL of ddH2O, fully reversing and mixing, and preparing a working solution: prepared 15 minutes before use, 8mLDetection antibodys + 32. mu.L Enzyme Concentrate, fully inverted and mixed evenly; (5) washing the plate: adding 300 mu L of Wash Buffer into each hole, soaking for 1 minute, pouring out the liquid in the 96-hole plate, properly beating on absorbent paper, drying the water, and repeatedly washing the plate for 5 times, or washing the plate by using a plate washing machine; (6) adding 100 mu L of prepared working solution into each hole, sticking a sealing plate film, and incubating for 1 hour in a constant temperature box at 37 ℃; (7) washing the plate according to the method of the step 5, wherein the plate is washed 7 times; (8) add 100. mu.L "TMB One-Step Substrate Reagent" into each well, incubate 30 minutes at room temperature in the dark; (9) when the liquid in the hole turns to dark blue, 50 μ L of "Stop Solution" is added to each hole, and the liquid turns from blue to yellow; (10) determining OD value at 450nm on a microplate reader within 5 minutes; (11) and (3) making a standard curve according to the concentration and the OD value of the standard substance, then calculating the sample concentration according to a standard curve equation, and converting the unit of the myocardial tissue sample into pg/mg according to the protein concentration measured before.

As shown in FIG. 5, on the 3 rd day after the operation, the contents of IL-6 and IL-1 beta in the serum and myocardial tissues of mice in Saline group and GLP-2 group are obviously higher than those in sham group, and the contents of IL-6 and IL-1 beta in the serum and myocardial tissues of mice in GLP-2 group are obviously lower than those in Saline group, which suggests that the teduotide can obviously reduce the circulating and IL-6 and IL-1 beta proinflammatory factors in myocardial tissues after myocardial ischemia-reperfusion.

Example 5 treatment with teduglutide significantly reduced neutrophil and macrophage infiltration in damaged myocardium following myocardial ischemia reperfusion

Materials: the small animal respirator, the animal operation illuminating lamp and the gas anesthesia system are purchased from Shanghai Yuyan scientific instruments, Inc., the operation microscopic instruments are purchased from Zhen Mei medical instruments, Inc., and the 4-0/7-0 suture line is purchased from Ningbo medical suture needle, Inc. Flow staining buffer, mouse CD11b-FITC monoclonal antibody was purchased from BD Biosciences, USA. Mouse Ly6G-PE, Ly6C-APC, F4/80-PerCy5.5 monoclonal antibody purchased from EBioscience. GentleMeC was purchased from Disociator Miltenyi. BD FACS Aria II flow cytometer was purchased from BD Biosciences.

And (3) leaving and taking a heart specimen: male C57BL/6 mice, 8 weeks old, were randomly assigned to sham (sham), Saline (Saline), and teduglutide (600. mu.g/kg GLP-2) groups, 5 each. Mouse myocardial ischemia reperfusion model was constructed as described above, and sham groups were threaded with only thread and not ligated. The teduglutide group was injected subcutaneously with 600 μ g/kg of teduglutide resistant to degradation by DPP4 immediately after reperfusion, twice daily for 3 consecutive days, the saline group was given an equal volume of saline at the same time, and mice were sacrificed 3 days after surgery to leave heart and blood specimens. After a mouse is anesthetized by isoflurane, the mouse is killed by a cervical dislocation method, then the mouse is fixed on a mouse plate, KCl (2mol/L)1mL is injected into a heart cavity after chest opening is rapidly carried out, the heart of the mouse stops jumping in a diastole, a small opening is cut in a right atrium, normal saline is injected from a left ventricle to carry out whole body perfusion, then the heart is cut off from the root of an aorta carefully, the blood stain on the surface of the heart of the mouse is washed by the normal saline, and gauze is wiped clean. The atrial and right ventricle portions were subtracted and the ischemic apical area was placed in a 1.5mL EP tube containing ice PBS.

Flow cytometry of myocardial tissues: (1) placing the ischemic apical area in a 1.5mL EP tube containing ice PBS, and cutting the tissue into fragments with scissors; (2) using genetlemecS^TMThe disasotor machine homogenized the tissue into a single cell suspension (large pieces of tissue were essentially not visible); (3) filter through nylon cloth to remove large tissue masses. Centrifuging at 300g and 4 ℃ for 5 minutes, discarding the supernatant, and resuspending the cells with 100. mu.L of staining buffer; (4) adding appropriate volume of cell surface marker conjugate antibody (CD11b-FITC, Ly6G-PE, Ly6C-APC, F4/80-PerCy5.5) or isotype control according to the antibody instruction, incubating at 4 deg.C in the dark for 30 min,can be placed on a rotator to rotate for sufficient incubation; (5) adding 1mL of staining buffer solution to wash the cells, centrifuging at 300g and 4 ℃ for 5 minutes, and removing supernatant; (6) after washing off redundant flow antibodies, loading the cells immediately after resuspending with 200 μ L of staining buffer solution, and detecting the expression condition of the surface markers by a flow cytometer; (7) CD11b⁺Ly6G⁺Labeling of neutrophils, CD11b⁺F4/80⁺Labeled macrophages, CD11b⁺F4/80⁺Ly6c^highMarking of proinflammatory macrophages, CD11b⁺F4/80⁺Ly6clow marks the suppressor macrophages.

As shown in fig. 6, on the 3 rd day after the operation, the proportion of neutrophils and macrophages in the myocardial tissues of mice in the salt group and the GLP-2 group is obviously higher than that of the sham group, and the proportion of neutrophils and macrophages in the heart of mice in the GLP-2 group is obviously lower than that of the salt group, which indicates that the teduotide can obviously reduce the infiltration amount of neutrophils and macrophages, and further analyzes the proportion of proinflammatory macrophages and anti-inflammatory macrophages, and the results show that: the proportion of proinflammatory macrophages and inflammation-inhibiting macrophages of mice in the GLP-2 group is obviously lower than that of mice in the Saline group.

Example 6 treatment with teduglutide significantly ameliorates intestinal flora disturbances following myocardial ischemia reperfusion

Materials: small animal ventilators, animal surgery lights, and gas anesthesia systems were purchased from Shanghai Yuyan scientific instruments, Inc. The surgical micro-instrument is purchased from Zhen Mei medical instruments Co. 4-0/7-0 suture was purchased from Ningbo medical needles, Inc. QIAamp DNA pool Mini Kit was purchased from Qiagen.

Collecting a fecal specimen: male C57BL/6 mice at 8 weeks of age were randomly divided into sham (sham), Saline (salt), and teduglutide (600. mu.g/kg GLP-2) groups, 5 sham, 6 Saline and GLP-2 groups, respectively. Mouse myocardial ischemia reperfusion model was constructed as described above, and sham groups were threaded with only thread and not ligated. The teduglutide group was injected subcutaneously with 600 μ g/kg of teduglutide resistant to degradation by DPP4, twice daily for 3 consecutive days immediately after reperfusion. The saline group was given an equal volume of saline at the same time. Mice were sacrificed 3 days post-surgery for sampling of mouse fecal specimens. Killing the mouse by breaking the neck, opening the abdominal cavity, shearing the terminal ileum and the cecum of the mouse by using sterile scissors, removing the head tip of a blue gun of a 1ml liquid-transferring gun sterilized at high temperature and high pressure by using the sterile scissors, and sucking excrement of the terminal ileum and the cecum into a 1.5ml enzyme-free sterile freezing tube by using the 1ml liquid-transferring gun, wherein each part is about 200 mg; and then immediately placing the freezing tube in liquid nitrogen for quick freezing, and then placing the tube in a refrigerator at the temperature of 80 ℃ below zero for storage for later use.

Extracting DNA of excrement: a commercial Kit QIAamp DNA Stool Mini Kit is adopted to extract genome DNA in a mouse excrement specimen, and the specific experimental flow is as follows: (1) taking out a standby excrement specimen from a refrigerator at the temperature of-80 ℃, placing the specimen on ice, transferring the specimen to an experimental table, and transferring the mouse excrement specimen from a freezing tube to a 2ml sterile enzyme-free centrifuge tube; (2) adding 1.6mL of BufferASL into the centrifuge tube; fully swirling the centrifuge tube until the sample is completely mixed uniformly, and centrifuging at 20000g/min, 4 ℃ for 2 min; (3) aspirate 1.2mL of supernatant, transfer to another 2mL centrifuge tube, and discard the fecal pellet. (4) Adding 1 inhibitor tablet into a centrifuge tube, and fully swirling until the inhibitor tablet is completely dissolved; standing and incubating the centrifugal tube at room temperature for 2min, and centrifuging at 20000g/min, 4 deg.C for 5 min; (5) transferring all the supernatant to a new 1.5mL centrifuge tube and centrifuging at 20000g/min, 4 ℃ for 5 min; (6) adding 25 ul of Proteinase K into a new 2mL centrifuge tube, and sucking 600 ul of the sample supernatant from step 5 into the centrifuge tube; (7) adding 600 mu l of buffer AL into the mixed solution of the sample supernatant and the protease K, fully and uniformly mixing, and incubating the centrifugal tube in a water bath at 70 ℃ for 1 hour; (8) then 600 mul of absolute ethyl alcohol is added into the centrifugal tube to be evenly stirred. (9) Adding 600 μ l of the above mixture into a spin column provided in the kit, centrifuging at 20000g/min, 4 deg.C for 1 min; the spin column was placed in a new 2ml collection tube and the tube containing the filtrate was discarded. (10) Adding 600 mul of absolute ethyl alcohol into the rotating column again, and repeating the step 9; (11) after the second washing is finished, repeating the step 10 for the third washing; (12) after the third washing, 500. mu.l of premixed Buffer AW1 was added to the spin column; centrifuge at 4 degree for 1min at 20000 g/min. The spin column was placed in a new collection tube and the tube containing the filtrate was discarded. (13) Adding 500 μ l premixed BuffeerAW 2 into spin column, centrifuging at 20000g/min, 4 deg.C for 4 min; discarding the tube containing the filtrate; (14) placing the spin column in a new 1.5mL centrifuge tube; the spin column lid was opened and 200. mu.l of BufferAE was pipetted directly onto the membrane; the lid was closed and incubated at room temperature for 1 min; centrifuging at 20000g/min for 1min at 4 ℃ to obtain purified DNA; (15) accurately quantifying the concentration of the sample by using a Qubit Fluorometer and a Qubit Fluorometer; the requirements of the Illumuna Miseq sequencing platform for genomic DNA are: the concentration is more than or equal to 5 ng/mu L, and the DNA amount is more than or equal to 30 ng.

Library construction and sequencing: according to the requirement of illumina high-throughput sequencing, bidirectional sequencing is carried out, and a target region and a fusion primer with a ' 5 ' linker-barcode-sequencing primer-specific primer-3 ' are designed. And a library is constructed by adopting a two-step PCR amplification method. The sequencing platform used in this study was Illumina Miseq2500, the sequencing type was PE250 primer sequences as follows:

F 5'-AATGATACGGCGACCACCGAGATCTACAC-

TCTTTCCCTACACGACGCTCTTCCGATCT-barcodeF 1-specific primer-3'

R 5'-CAAGCAGAAGACGGCATACGAGAT-barcode R2-

GTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-barcode R1-specific primer-3'

As shown in FIG. 7, the intestinal tract pathogenic bacteria Proteobacteria (Proteobacteria) -Gamma aproteobacteria (Proteobacteria) of the mice in the salt group are remarkably increased compared with the intestinal tract pathogenic bacteria Proteobacteria (Proteobacteria) of the mice in the sham group, and the relative abundance of the intestinal tract resident bacteria Bacillus (Bacteria) is remarkably reduced, while the intestinal tract pathogenic bacteria Proteobacteria (Proteobacteria) -Gamma aproteobacteria (Proteobacteria) of the GLP-2 group are reduced in limitation, so that the flora disturbance is improved.

Example 7 treatment with teduglutide significantly inhibited intestinal flora translocation following myocardial ischemia reperfusion

Materials: small animal ventilators, animal surgery lights, and gas anesthesia systems were purchased from Shanghai Yuyan scientific instruments, Inc. The surgical micro-instrument is purchased from Zhen Mei medical instruments Co. 4-0/7-0 suture was purchased from Ningbo medical needles, Inc.

And (3) detecting flora shift: male C57BL/6 mice at 8 weeks of age were randomized into sham groups(sham group), Saline group (Saline group), and teduglutide group (600. mu.g/kg GLP-2 group), 4 each. Mouse myocardial ischemia reperfusion model was constructed as described above, and sham groups were threaded with only thread and not ligated. The teduglutide group was injected subcutaneously with 600 μ g/kg of teduglutide resistant to degradation by DPP4, twice daily for 3 consecutive days immediately after reperfusion. The saline group was given an equal volume of saline at the same time. On the third day after operation, 5X 10 is used⁹And (3) intragastrically irrigating the colony forming unit with the bioluminescent element-labeled citrobacter, killing the mice after 6h, taking blood, mesenteric lymph nodes, spleen and heart homogenate supernatant under an aseptic condition, performing plate-laying culture for 24 hours, and detecting and counting the number of colonies with fluorescence by using an IVIS (in vivo imaging system) living body imager to evaluate whether the intestinal flora shifts and which organs the shifted flora enters.

As shown in FIG. 8, bacterial colonies were observed in blood, mesenteric lymph nodes, spleen and heart tissues of mice in the Saline group, and the number of colonies in blood, mesenteric lymph nodes, spleen and heart tissues of mice in the GLP-2 group was significantly reduced compared to that of the Saline group, suggesting that the replacement dolutesin treatment can effectively reduce the intestinal flora shift after myocardial ischemia reperfusion.

Claims

1. The application of the teduglutide in preparing the medicine for treating myocardial ischemia reperfusion injury is characterized by specifically preparing the medicine for treating intestinal flora shift and inflammatory storm caused by intestinal flora imbalance caused by myocardial ischemia reperfusion after acute myocardial infarction vessel recanalization.

2. The use of claim 1, wherein the myocardial ischemia-reperfusion-induced injury is further myocardial injury following revascularization after acute coronary occlusion.

3. The use of claim 2, wherein said further myocardial damage comprises oxidative stress damage, inflammatory response damage.

4. The use according to claim 1, wherein the inflammatory storm is a disturbance of intestinal flora resulting in intestinal permeability and barrier function disruption after myocardial ischemia-reperfusion, triggering migration of intestinal flora into circulation and LPS bleeding, promoting activation and mobilization of inflammatory cells such as neutrophils and monocytes macrophages, producing and secreting a number of inflammatory factors and chemokines, promoting infiltration of pro-inflammatory cells into damaged myocardium, resulting in activation and persistence of inflammatory response after myocardial ischemia-reperfusion.

5. The use of claims 1-4, wherein the amino acid sequence of teduglutide is as follows:

6. the use of claims 1-4, wherein the mice with teduglutide are administered twice daily at a dose of 600 and 1200 μ g/kg, preferably 600 μ g/kg.

7. The use of claims 1-4, wherein the human dose of teduglutide is 66-132 μ g/kg twice daily, preferably 66 μ g/kg per dose.

8. The use of claims 1-4, wherein the teduglutide can be in the form of its drug substance, solvate and salt, and other glucagon-like peptide-2 analogs with similar amino acid sequences.

9. The use of claim 8, wherein the glucagon-like peptide-2 analog is apraglutide and the specific amino acid sequence is:

His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-norleucine-D-Phenylalanine-Thr-Ile-Leu-Asp-Leu-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-NH₂。

10. the use of claims 1-4, wherein the teduglutide can be formulated with pharmaceutically acceptable conventional pharmaceutical excipients into a pharmaceutical formulation.

11. The use according to claim 10, wherein the pharmaceutical formulation is administered parenterally; the medicinal preparation is an injection preparation or a powder injection.