CN118121634B - Application of IMRC-Exo in the preparation of drugs to reduce multiple organ damage after cardiac arrest resuscitation and drugs - Google Patents

Abstract

The application provides an application of IMRC-Exo in preparing a medicament for relieving multi-organ injury after cardiac arrest and resuscitation and a medicament, and relates to the technical field of cell pharmaceutical preparations. According to the application, through researching the application effect of the IMRC-Exo in multi-organ injury treatment after cardiac arrest resuscitation, the IMRC-Exo can be obtained to effectively relieve multi-organ injury after cardiac arrest resuscitation. Specifically, the application obtains the IMRC-Exo group relative to the conventional cardiac arrest resuscitation animal model group by applying the pharmaceutical dosage to the cardiac arrest resuscitation animal model, and the IMRC-Exo group has statistical significance in the aspects of myocardial injury markers, brain injury markers, kidney and intestine injury markers, pro-inflammatory factor content analysis of multi-organ tissues and apoptosis degree analysis of the multi-organ tissues, thereby providing a new thought for researching and developing high-efficiency drugs for treating syndrome after cardiac arrest.

Description

Application of IMRC-Exo in the preparation of drugs to reduce multiple organ damage after cardiac arrest and resuscitation

Technical Field

The present invention relates to the technical field of cell medicine preparations, and in particular to an application of IMRC-Exo in the preparation of a medicine for alleviating multiple organ damage after cardiac arrest resuscitation and a medicine.

Background technique

Cardiac arrest has always been a major health problem with high incidence and mortality worldwide. Currently, efforts to improve cardiac arrest treatment strategies and continuously improve the clinical prognosis of such patients have become important tasks in this field.

Studies have shown that post-cardiac arrest syndrome is an important cause of death in patients with cardiac arrest after successful resuscitation. It is caused by systemic ischemia-reperfusion injury caused by cardiac arrest resuscitation, and clinically manifested as multi-organ system dysfunction with heart and brain organ damage as the main cause. At present, the therapeutic hypothermia and other means recommended by international guidelines are not enough to significantly improve the clinical treatment effect of post-cardiac arrest syndrome. In addition, some treatment methods proven to be effective in the laboratory have not been successfully transformed and applied in the clinic. It is urgent to explore new effective and clinically feasible methods for the treatment of post-cardiac arrest syndrome.

In view of this, the present invention is proposed.

Summary of the invention

Explanation of the abbreviations involved in this application:

MSC: Mesenchymal stem cells (MSC);

IMRC: Immunity and matrix regulatory cells (IMRC) formed by inducing differentiation of human embryonic stem cells with unlimited stable proliferation and totipotent differentiation capacity as seed cells;

Exo: Exosomes (Exo);

IMRC-Exo: refers to exosomes derived from immune and matrix regulatory cells (Human embryonic stem cells derived immune and matrix regulatory cells-exosome) formed by the directed differentiation of human embryonic stem cells, referred to as hESC-IMRC-Exo.

The purpose of the present invention is to confirm the application effect of IMRC-Exo in the treatment of multiple organ damage after cardiac arrest resuscitation through research, and on this basis, explore the research and development and clinical transformation of IMRC-Exo exosome drugs, which will be conducive to the development of new and efficient drugs for the treatment of post-cardiac arrest syndrome, and has important scientific research significance and clinical application prospects.

In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

The present invention provides an application of IMRC-Exo in preparing a drug for alleviating multiple organ damage after cardiac arrest resuscitation.

Furthermore, the application is to administer a pharmaceutical dose of IMRC-Exo.

Furthermore, the IMRC-Exo is secreted by IMRC cells whose cell surface markers CD105, CD73 and CD90 are all >95%, and whose immunogenicity-related protein expressions achieve HLA-DR <5%, HLA-E and HLA-G >50%.

Furthermore, the administration method is intravenous administration of 20-200 ml of an injection having a concentration of 5×10 ¹⁰ IMRC-Exo particles/mL.

Furthermore, the pharmaceutically effective dose of the IMRC-Exo is 2.5×10 ¹⁰ -2.5×10 ¹¹ IMRC-Exo particles/kg.

Furthermore, the multiple organ damage after cardiac arrest resuscitation includes:

Heart function damage after cardiac arrest resuscitation, brain function damage after cardiac arrest resuscitation, renal function damage after cardiac arrest resuscitation, and intestinal function damage after cardiac arrest resuscitation.

The present invention provides a medicine for alleviating multiple organ damage after cardiac arrest resuscitation. The active components of the medicine include IMRC-Exo and pharmaceutically acceptable excipients.

Furthermore, the content of IMRC-Exo in the drug is 1×10 ¹² to 1×10 ¹³ particles.

Furthermore, the pharmaceutically acceptable excipients include one or more of a diluent, a binder, a wetting agent, a disintegrant, a lubricant, a solubilizer, a pH regulator and an osmotic pressure regulator.

Furthermore, the dosage form of the drug is an injection;

Preferably, the administration route of the injection includes one of intravenous injection, intraperitoneal injection, intramuscular injection or subcutaneous injection, preferably intravenous injection.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention provides an application of IMRC-Exo in the preparation of a drug for reducing multiple organ damage after cardiac arrest and resuscitation. This application confirms the application effect of IMRC-Exo in the treatment of multiple organ damage after cardiac arrest and resuscitation through research, and on this basis explores the research and development and clinical transformation of IMRC-Exo exosome drugs, which has important scientific research significance for the development of new and efficient drugs for the treatment of post-cardiac arrest syndrome.

The present invention provides a drug for alleviating multiple organ damage after cardiac arrest resuscitation, wherein the active components of the drug include IMRC-Exo and pharmaceutically acceptable excipients. Experiments have shown that IMRC-Exo at a pharmaceutically effective dose can effectively alleviate multiple organ damage after cardiac arrest resuscitation.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

FIG1a is a graph showing changes in stroke volume (SV) of animals in the Sham, CPR, and CPR+IMRC-Exo groups provided in Example 2 of the present invention;

FIG1b is a graph showing changes in global ejection fraction (GEF) of animals in the Sham, CPR, and CPR+IMRC-Exo groups provided in Example 2 of the present invention;

FIG1c is a graph showing changes in cardiac troponin (cTnI) in animals in the Sham, CPR, and CPR+IMRC-Exo groups provided in Example 2 of the present invention;

FIG2a is a graph showing changes in neuron-specific enolase (NSE) in animals in the Sham, CPR, and CPR+IMRC-Exo groups provided in Example 2 of the present invention;

FIG2 b is a graph showing changes in neurological deficit scores (NDS) of animals in the Sham, CPR, and CPR+IMRC-Exo groups provided in Example 2 of the present invention;

FIG2c is a graph showing changes in brain function performance grade (CPC) of animals in the Sham, CPR, and CPR+IMRC-Exo groups provided in Example 2 of the present invention;

FIG3a is a graph showing changes in creatinine (Cr) in animals in the Sham, CPR, and CPR+IMRC-Exo groups provided in Example 2 of the present invention;

FIG3 b is a graph showing changes in intestinal fatty acid binding protein (IFABP) in animals in the Sham, CPR, and CPR+IMRC-Exo groups provided in Example 2 of the present invention;

FIG4a is a staining diagram of the degree of apoptosis in multiple organ tissues of animals in the Sham, CPR, and CPR+IMRC-Exo groups 24 hours after resuscitation provided in Example 2 of the present invention;

FIG4b is a bar graph showing the degree of apoptosis in multiple organ tissues of animals in the Sham, CPR and CPR+IMRC-Exo groups 24 hours after resuscitation provided in Example 2 of the present invention;

FIG5a is a graph showing the content analysis of tumor necrosis factor-a (TNF-a) in multiple organ tissues of animals in the Sham, CPR and CPR+IMRC-Exo groups 24 hours after resuscitation provided in Example 2 of the present invention;

FIG5 b is a graph showing the interleukin-1β (IL-1β) content analysis of multiple organ tissues of the Sham, CPR, and CPR+IMRC-Exo group animals 24 hours after resuscitation provided in Example 2 of the present invention.

Detailed ways

The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

According to one aspect of the present invention, a use of IMRC-Exo in the preparation of a drug for reducing multiple organ damage after cardiac arrest resuscitation.

The present invention provides an application of IMRC-Exo in the preparation of a drug for reducing multiple organ damage after cardiac arrest and resuscitation. This application confirms the application effect of IMRC-Exo in the treatment of multiple organ damage after cardiac arrest and resuscitation through research, and on this basis explores the research and development and clinical transformation of IMRC-Exo exosome drugs, which has important scientific research significance for the development of new and efficient drugs for the treatment of post-cardiac arrest syndrome.

It should be noted that in recent years, studies have shown that mesenchymal stem cells (MSC) have a variety of biological effects such as anti-inflammatory, antioxidant, and anti-apoptotic. In addition, studies have also found that the use of human embryonic stem cells with unlimited stable proliferation and omnipotent differentiation ability as seed cells to induce differentiation to form immune and matrix regulatory cells (IMRC) is superior to MSC from traditional adult tissues such as bone marrow, umbilical cord, and fat in terms of cell quality, immune regulation, and damage repair ability, thus becoming a more advantageous stem cell therapy. In addition, exosomes (Exo) are important mediators for MSC treatment of various acute diseases. They have various biological effects of MSC and are easier to obtain, store, and safer than MSC, thus becoming a good alternative to MSC treatment. However, there has been no relevant research report on the study of IMRC-Exo in the preparation of drugs to reduce multi-organ damage after cardiac arrest resuscitation.

In a preferred embodiment of the present invention, the use comprises administering a pharmaceutical dose of IMRC-Exo, wherein:

The administration method is to intravenously administer an equal amount of IMRC-Exo dissolved in a solvent;

The pharmaceutically effective dose of the IMRC-Exo is 2.5×10 ¹⁰ -2.5×10 ¹¹ IMRC-Exo particles/kg.

In a preferred embodiment of the present invention, the IMRC-Exo is secreted by IMRC cells whose cell surface markers CD105, CD73 and CD90 are all >95%, and whose immunogenicity-related protein expressions achieve HLA-DR <5%, HLA-E and HLA-G >50%.

Preferably, the preparation process of IMRC-Exo is as follows:

1) Human embryonic stem cells were used to induce differentiation into IMRCs with a purity of >95%. The expression of IMRC cell surface markers CD105, CD73 and CD90 were controlled to be >95%, and the expression of immunogenicity-related proteins was controlled to achieve HLA-DR <5%, HLA-E and HLA-G >50%, thereby obtaining standardized, low-immunogenic IMRCs;

2) Use tangential flow, ultrafiltration, ultracentrifugation, chromatography, immunoaffinity and other methods to extract Exo from IMRC, and then obtain IMRC-Exo of standard quality through purification, identification, counting, specific membrane protein expression and morphological observation.

In a preferred embodiment of the present invention, the multiple organ damage after cardiac arrest resuscitation includes:

Heart function damage after cardiac arrest resuscitation, brain function damage after cardiac arrest resuscitation, renal function damage after cardiac arrest resuscitation, and intestinal function damage after cardiac arrest resuscitation.

According to one aspect of the present invention, a drug for reducing multiple organ damage after cardiac arrest resuscitation is provided, wherein the active ingredients of the drug include IMRC-Exo and pharmaceutically acceptable excipients.

The present invention provides a drug for alleviating multiple organ damage after cardiac arrest resuscitation, wherein the active components of the drug include IMRC-Exo and pharmaceutically acceptable excipients. Experiments have shown that IMRC-Exo at a pharmaceutically effective dose can effectively alleviate multiple organ damage after cardiac arrest resuscitation.

In a preferred embodiment of the present invention, the content of IMRC-Exo in the drug is 1×10 ¹² to 1×10 ¹³ particles.

Preferably, the content of IMRC-Exo in every 20 ml of the drug is 1×10 ¹² IMRC-Exo particles;

It should be noted that the drug of the present application needs to contain a pharmaceutically effective dose of IMRC-Exo particles, and the pharmaceutically effective dose of IMRC-Exo is 2.5×10 ¹⁰ ~2.5×10 ¹¹ IMRC-Exo particles/kg. For example, for an individual weighing 35-40 kg, in the intravenous administration method, 20-200 ml of a preparation with a concentration of 5×10 ¹⁰ IMRC-Exo particles/mL needs to be administered.

In the above preferred embodiment, the pharmaceutically acceptable excipients include one or more of a diluent, a binder, a wetting agent, a disintegrant, a lubricant, a solubilizer, a pH regulator and an osmotic pressure regulator.

In a preferred embodiment of the present invention, the dosage form of the drug is an injection;

Preferably, the administration route of the injection includes one of intravenous injection, intraperitoneal injection, intramuscular injection or subcutaneous injection, preferably intravenous injection.

The technical solution of the present invention will be further described below in conjunction with embodiments.

Example 1

1. Animal preparation:

1. Before the experiment, fast for 12 hours but do not drink water.

2. On the morning of the experiment, anesthesia was induced by intramuscular injection of tiletamine/zolazepam 5 mg/kg and thiazine 1 mg/kg, and general anesthesia was maintained by intravenous injection of propofol 2 mg/kg and intravenous infusion of propofol 4 mg/kg/h. At the same time, butorphanol 8 μg/kg intravenous push and 3 μg/kg/h intravenous infusion were used for analgesia.

3. Orotracheal intubation was performed and the end-tidal carbon dioxide partial pressure (ETCO ₂ ) monitoring device was connected to the ventilator. The ventilation parameters of the latter were set to volume control mode, tidal volume 10 ml/kg, oxygen concentration 21%, and the initial ETCO ₂ was maintained in the normal range of 35-40 mmHg by adjusting the respiratory rate.

4. Cut the skin under direct vision, peel off the subcutaneous fascia and muscles, expose the bilateral femoral arteries and veins, insert the arterial catheter of the pulse indicator continuous cardiac output (PiCCO) monitor into the left femoral artery, and insert the pressure monitoring catheter into the thoracic aorta and right atrium of the right femoral artery and vein respectively. In addition, cut the skin under direct vision, peel off the above tissues to expose the right internal and external jugular veins, and insert the ventricular fibrillation induction electrode and the venous catheter of the PiCCO monitor respectively.

5. Use a temperature-control blanket to maintain a normal body temperature of around 38°C throughout the process.

2. Model establishment:

1. Condition setting of cardiac arrest resuscitation model: cardiac arrest for 12 min + cardiopulmonary resuscitation for 6 min.

2. Method of inducing cardiac arrest: The ventricular fibrillation induction electrode is implanted through the right ventricle, connected to a power source and releases 1 mA alternating current to induce ventricular fibrillation. After confirmation of success, observe for 12 minutes without intervention.

3. Methods of cardiopulmonary resuscitation:

1) Mechanical chest compressions, with a depth of 5 cm and a frequency of 100 times/min;

2) ventilator-assisted ventilation, parameters were volume control mode, tidal volume 7 ml/kg, oxygen concentration 100%, and respiratory rate 10 times/min;

3) Epinephrine: 20 μg/kg intravenous injection at 2 minutes after cardiopulmonary resuscitation, and then repeated every 3 minutes;

4) Defibrillation: 150J defibrillation was performed once every 6 minutes of CPR;

5) If spontaneous circulation has not been restored, immediately restart cardiopulmonary resuscitation and perform defibrillation once after 2 minutes. Repeat this cycle until resuscitation is successful or declare resuscitation a failure after a maximum of 5 times.

4. Post-resuscitation monitoring method: 1) Continue anesthesia monitoring; 2) Restart mechanical ventilation with the same parameters as before, i.e., volume control mode, tidal volume 10 ml/kg, oxygen concentration 21%, and respiratory rate restored to the state before modeling); 3) Continue monitoring for 6 hours.

5. Post-resuscitation observation method: After the animal monitoring is completed, disinfect and suture all incisions, take the animal offline and remove the endotracheal tube, send the animal to the pigsty, and continue to observe for 18 hours.

Example 2

1. Animal randomization and intervention:

Experimental groups: 18 domestic healthy male white pigs, weighing 35-40 kg, were randomly divided into 3 groups: sham operation (Sham) group, cardiopulmonary resuscitation (CPR) group, and CPR+IMRC-Exo group, with 6 pigs in each group.

Interventions:

1) Sham group: No pig cardiac arrest resuscitation model was established, and the same amount of solvent was given as in other groups at the same time.

2) CPR group: A pig cardiac arrest resuscitation model was established, and 5 minutes after successful resuscitation, the same amount of solvent was given as in other groups at the same time.

3) CPR+IMRC-Exo group: A pig cardiac arrest resuscitation model was established, and 5 minutes after successful resuscitation, an equal amount of solvent-prepared IMRC-Exo was applied through the internal jugular vein, with a total number of 1× ¹⁰¹² particles.

Note: The solvents for the three groups were all 20 ml of normal saline, which was pumped into the internal jugular vein within 30 minutes.

(II) Observation indicators:

1. Before modeling, record the body weight, heart rate, blood pressure, _ETCO2 and other values of the three groups of animals, and detect the results of arterial blood gas analysis.

2. During cardiopulmonary resuscitation, the changes in the levels of aortic blood pressure and right atrial pressure were continuously monitored, and the value of coronary perfusion pressure was calculated by the difference between the two. The cardiopulmonary resuscitation duration, epinephrine dosage, number of defibrillations, resuscitation success rate and other results of the three groups of animals were also recorded.

3. Before modeling and 1h, 2h, 4h, and 6h after resuscitation, the PiCCO monitor was used to regularly evaluate changes in cardiac function indicators such as stroke volume (SV) and global ejection fraction (GEF).

4. Before modeling and 1h, 2h, 4h, 6h, and 24h after resuscitation, collect 2 ml of venous blood samples, centrifuge to obtain plasma, freeze in a -80℃ deep-freezer, and use enzyme-linked immunosorbent assay to test at a later date:

Myocardial injury marker - cardiac troponin I (cTnI);

Brain injury marker - neuron-specific enolase (NSE);

Renal injury markers - creatinine (Cr);

Serum levels of intestinal fatty acid binding protein (iFABP), a marker of intestinal injury.

5. At 24 hours after resuscitation, the neurological deficit score (NDS) and cerebral performance grade (CPC) were used to evaluate the neurological status of the three groups of animals.

6. At 24 hours after resuscitation, once all the above samples and data collection were completed, the animals were euthanized and tissues from the left ventricular apex, cerebral cortex, hippocampus, upper pole of right kidney, terminal ileum and other parts were quickly obtained. Some of the tissues were fixed, embedded, and sliced to prepare pathological specimens. The TUNEL method (TUNEL) was used to detect the apoptosis index of the heart, brain, kidney, intestine and other organ tissues at an appropriate time.

In addition, some fresh tissues were frozen in a -80°C deep freezer, and the levels of proinflammatory factors - tumor necrosis factor-a (TNF-a) and interleukin-1β (IL-1β) in various organ tissues were detected by enzyme-linked immunosorbent assay at an appropriate time.

3. Research results:

1. Baseline status of the three groups of animals before modeling:

Before modeling, there were no significant differences in basic physiological indicators such as body weight, heart rate, mean arterial pressure, ETCO ₂ , pH value, PO ₂ , PCO ₂ , and lactic acid among the three groups of animals (all P > 0.05). See Table 1.

Table 1. Basic information of the three groups of animals ( ±s):

Note: ETCO ₂ , end-tidal carbon dioxide partial pressure; PO ₂ , partial pressure of oxygen; PCO ₂ , partial pressure of carbon dioxide; Sham, sham operation; CPR, cardiopulmonary resuscitation; IMRC-Exo, immune and stromal regulatory cell-derived exosomes.

2. Effects and outcomes of cardiopulmonary resuscitation in the two groups of animals during modeling:

During the modeling period, the experimental animal model was established in the CPR group and the CPR+IMRC-Exo group using the parameters of cardiac arrest for 12 minutes and cardiopulmonary resuscitation for 6 minutes.

The data showed that during CPR, the coronary perfusion pressure of the two groups of animals maintained the same trend of change and was maintained at a roughly consistent level, with no statistically significant difference between the two groups (all P > 0.05). As a result, there were no statistically significant differences in the CPR duration, adrenaline dosage, number of defibrillators, and resuscitation success rate between the two groups (all P > 0.05). See Table 2.

Table 2. Cardiopulmonary resuscitation effects and outcomes of the two groups of animals ( ±s):

Note: CPR, cardiopulmonary resuscitation; IMRC-Exo, immune and matrix regulatory cell-derived exosomes. The data of animals that failed resuscitation in the CPR group and CPR+IMRC-Exo group were only used for the analysis of cardiopulmonary resuscitation effect and outcome, and no data were used for the analysis of other indicators.

3. Changes of cardiac function indexes and myocardial injury markers in the three groups of animals before and after modeling:

FIG. 1a to FIG. 1c are diagrams showing changes in cardiac function indices and myocardial injury markers in animals in the Sham, CPR, and CPR+IMRC-Exo groups provided in this embodiment, wherein:

FIG1a is a graph showing changes in stroke volume (SV) of animals in the Sham, CPR, and CPR+IMRC-Exo groups provided in this example;

FIG1b is a graph showing changes in global ejection fraction (GEF) of animals in the Sham, CPR, and CPR+IMRC-Exo groups provided in this example;

FIG1c is a graph showing changes in cardiac troponin (cTnI) in animals in the Sham, CPR, and CPR+IMRC-Exo groups provided in this example;

In Figure 1a to Figure 1c: BL, baseline; Sham, sham operation; CPR, cardiopulmonary resuscitation; IMRC-Exo, exosomes derived from immune and matrix regulatory cells; As can be seen from Figure 1a to Figure 1c, compared with the Sham group, the CPR group and the CPR+IMRC-Exo group, ^* P <0.05; compared with the CPR group, the CPR+IMRC-Exo group, ^# P <0.05.

Before modeling, there were no significant differences in the baseline levels of cardiac function indexes SV and GEF and myocardial injury marker cTnI among the three groups (all P > 0.05). After resuscitation, compared with the Sham group, the SV and GEF values of the CPR group and the CPR+IMRC-Exo group were significantly reduced, and the differences among the groups were statistically significant (all P < 0.05).

However, the SV values at 1 and 2 h after resuscitation, as well as the GEF values at each time point after resuscitation in the CPR+IMRC-Exo group were significantly higher than those in the CPR group, and the differences between the groups were statistically significant (all P < 0.05).

In addition, compared with the Sham group, the serum levels of cTnI in the CPR group and CPR+IMRC-Exo group were significantly increased at each time point after resuscitation, and the differences between the groups were statistically significant (all P < 0.05).

However, the serum levels of cTnI in the CPR+IMRC-Exo group were significantly lower than those in the CPR group 2 h after resuscitation, and the differences between the groups were statistically significant (all P < 0.05). See Figure 1a to Figure 1c for details.

4. Changes of brain injury markers and neurological function indicators in the three groups of animals before and after modeling:

FIG. 2a to FIG. 2c are graphs showing changes in brain injury markers and neurological function indicators in the Sham, CPR, and CPR+IMRC-Exo groups of animals provided in this example, wherein:

FIG2a is a graph showing changes in neuron-specific enolase (NSE) in animals in the Sham, CPR, and CPR+IMRC-Exo groups provided in this example;

FIG2 b is a graph showing changes in neurological deficit scores (NDS) of animals in the Sham, CPR, and CPR+IMRC-Exo groups provided in this example;

FIG2c is a graph showing changes in brain function performance grade (CPC) of animals in the Sham, CPR, and CPR+IMRC-Exo groups provided in this example;

In Figure 2a to Figure 2c: BL, baseline; Sham, sham operation; CPR, cardiopulmonary resuscitation; IMRC-Exo, exosomes derived from immune and matrix regulatory cells; As can be seen from Figure 2a to Figure 2c, compared with the Sham group, the CPR group and the CPR+IMRC-Exo group, ^* P <0.05; compared with the CPR group, the CPR+IMRC-Exo group, ^# P <0.05.

Before modeling, there was no significant difference in the serum levels of NSE, a marker of brain injury, among the three groups of animals (all P > 0.05).

After resuscitation, compared with the Sham group, the serum NSE levels of the animals in the CPR group and the CPR+IMRC-Exo group at each time point were significantly increased, and the differences between the groups were statistically significant (all P < 0.05). However, the serum NSE levels of the animals in the CPR+IMRC-Exo group were significantly lower than those in the CPR group 4 hours after resuscitation, and the differences between the groups were statistically significant (all P < 0.05).

In addition, compared with the Sham group, the neurological function indexes NDS and CPC scores of the animals in the CPR group and CPR+IMRC-Exo group at 24 h after resuscitation were significantly increased, and the differences between the groups were statistically significant (all P < 0.05).

However, the NDS and CPC scores of the CPR+IMRC-Exo group were significantly lower than those of the CPR group at 24 h after resuscitation, and the differences between the groups were statistically significant (all P < 0.05). See Figure 2a to Figure 2c for details.

5. Changes of renal and intestinal injury markers in the three groups of animals before and after modeling

FIG. 3a to FIG. 3b are graphs showing changes in renal and intestinal injury markers in the Sham, CPR, and CPR+IMRC-Exo groups of animals provided in this example, wherein:

FIG3a is a graph showing changes in creatinine (Cr) in animals in the Sham, CPR, and CPR+IMRC-Exo groups provided in this example;

FIG3 b is a graph showing changes in intestinal fatty acid binding protein (IFABP) in animals in the Sham, CPR, and CPR+IMRC-Exo groups provided in this example;

In Figure 3a and Figure 3b: BL, baseline; Sham, sham operation; CPR, cardiopulmonary resuscitation; IMRC-Exo, exosomes derived from immune and matrix regulatory cells; As can be seen from Figure 3a and Figure 3b, compared with the Sham group, the CPR group and the CPR+IMRC-Exo group, ^* P <0.05; compared with the CPR group, the CPR+IMRC-Exo group, ^# P <0.05.

As shown in Figures 3a and 3b, before modeling, there were no significant differences in the serum levels of Cr and IFABP, the markers of renal and intestinal injury, among the three groups of animals (all P > 0.05). After resuscitation, compared with the Sham group, the serum levels of Cr and IFABP in the CPR group at each time point were significantly increased, and the serum levels of Cr at each time point and the serum levels of IFABP after 2 h of resuscitation in the CPR+IMRC-Exo group were significantly increased, and the differences between the groups were statistically significant (all P < 0.05). However, the serum levels of Cr and IFABP in the CPR+IMRC-Exo group after 2 h of resuscitation were significantly lower than those in the CPR group, and the differences between the groups were statistically significant (all P < 0.05).

6. Analysis of the degree of cell apoptosis in multiple organ tissues of the three groups of animals 24 hours after resuscitation

FIG. 4a and FIG. 4b are diagrams for analyzing the degree of apoptosis in multiple organ tissues of animals in the Sham, CPR, and CPR+IMRC-Exo groups 24 hours after resuscitation provided in this example.

FIG4a is a staining diagram of the degree of apoptosis in multiple organ tissues of animals in the Sham, CPR, and CPR+IMRC-Exo groups provided in this example 24 hours after resuscitation;

FIG4b is a bar graph showing the degree of apoptosis in multiple organ tissues of animals in the Sham, CPR, and CPR+IMRC-Exo groups 24 hours after resuscitation provided in this example;

In Figure 4a and Figure 4b: Sham, sham operation; CPR, cardiopulmonary resuscitation; IMRC-Exo, exosomes derived from immune and matrix regulatory cells; As can be seen from Figure 4a and Figure 4b, compared with the Sham group, the CPR group and the CPR+IMRC-Exo group, ^* P <0.05; compared with the CPR group, the CPR+IMRC-Exo group, ^# P <0.05.

As shown in Figures 4a and 4b, at 24 hours after resuscitation, the animals in each group were euthanized, and then some tissues such as the left ventricular apex, cerebral cortex, hippocampus, upper pole of the right kidney, and terminal ileum were quickly obtained for pathological examination and analysis. The results showed that compared with the Sham group, the apoptosis index of the heart, brain, kidney, intestine and other organ tissues of the CPR group and the CPR+IMRC-Exo group was significantly increased, and the differences between the groups were statistically significant (all P <0.05). However, the apoptosis index of the above-mentioned multiple organ tissues of the CPR+IMRC-Exo group was significantly lower than that of the CPR group, and the differences between the groups were statistically significant (all P <0.05).

7. Analysis of pro-inflammatory factors in multiple organ tissues of the three groups of animals 24 hours after resuscitation

FIG. 5a and FIG. 5b are graphs for analyzing the contents of pro-inflammatory factors in multiple organ tissues of animals in the Sham, CPR, and CPR+IMRC-Exo groups 24 hours after resuscitation provided in this example.

FIG5a is a graph showing the content analysis of tumor necrosis factor-a (TNF-a) in multiple organ tissues of animals in the Sham, CPR, and CPR+IMRC-Exo groups at 24 hours after resuscitation provided in this example;

FIG5 b is a graph showing the content analysis of interleukin-1β (IL-1β) in multiple organ tissues of the Sham, CPR, and CPR+IMRC-Exo group animals at 24 hours after resuscitation provided in this example;

In Figure 5a and Figure 5b: Sham, sham operation; CPR, cardiopulmonary resuscitation; IMRC-Exo, exosomes derived from immune and matrix regulatory cells; As can be seen from Figure 5a and Figure 5b, compared with the Sham group, the CPR group and the CPR+IMRC-Exo group, ^* P <0.05; compared with the CPR group, the CPR+IMRC-Exo group, ^# P <0.05.

As shown in Figures 5a and 5b, 24 hours after resuscitation, the animals were killed as above and samples of heart, brain, kidney, intestine and other organ tissues were obtained for detection and analysis of the content of proinflammatory factors. The results showed that compared with the Sham group, the content of proinflammatory factors TNF-a and IL-1β in the heart, brain, kidney, intestine and other organ tissues of animals in the CPR group and CPR+IMRC-Exo group was significantly increased, and the differences between the groups were statistically significant (all P <0.05). However, the content of proinflammatory factors in the above-mentioned multiple organ tissues of animals in the CPR+IMRC-Exo group was significantly lower than that in the CPR group, and the differences between the groups were statistically significant (all P <0.05).

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims (5)

1. Application of IMRC-Exo in preparing medicines for relieving multi-organ injury after cardiac arrest resuscitation;

   The multi-organ injury after cardiac arrest resuscitation is as follows: cardiac function injury following cardiac arrest resuscitation, brain function injury following cardiac arrest resuscitation, renal function injury following cardiac arrest resuscitation, and intestinal function injury following cardiac arrest resuscitation;

   The IMRC-Exo refers to immune and matrix regulating cell-derived exosomes formed by directional induced differentiation of human embryonic stem cells.
2. 2. The use according to claim 1, wherein the use is the administration of a pharmaceutical dose of IMRC-Exo.
3. 3. The use according to claim 1, wherein the IMRC-Exo is secreted by IMRC cells with cell surface markers CD105, CD73 and CD90 of >95% and immunogenicity related protein expression achieving HLA-DR <5%, HLA-E and HLA-G > 50%.
4. 4. The use according to claim 2, wherein the administration is by intravenous administration of 20-200mL of the formulation at a concentration of 5 x 10 ¹⁰ IMRC-Exo particles/mL.
5. 5. The use according to claim 2, wherein the pharmaceutically effective dose of IMRC-Exo is 2.5 x 10 ¹⁰~2.5×10¹¹ IMRC-Exo particles/kg.