Application of IMRC-Exo in preparation of medicine for relieving limb local muscle injury after agkistrodon acutus bites and medicine

Abstract

The invention provides application of IMRC-Exo in preparation of a medicine for relieving limb local muscle injury after agkistrodon acutus bites and the medicine, and relates to the technical field of snake venom bites. According to the application, the application effect of the IMRC-Exo in treatment of limb injury after agkistrodon acutus bites is confirmed through research, and research and development and clinical conversion of exosome drugs of the IMRC-Exo are explored on the basis of the application effect of the IMRC-Exo in treatment of limb injury after agkistrodon acutus bites. Specifically, an IMRC-Exo preparation is applied to the periphery of a bite muscle tissue of an agkistrodon acutus bite animal model, and compared with the agkistrodon acutus bite animal model, the IMRC-Exo treatment can effectively reduce the peripheral diameter of an animal limb after agkistrodon acutus bite and the level of a muscle injury marker in serum; and the effect improvement has statistical significance in analysis of the pathological injury degree, the muscle tissue cell apoptosis degree, the muscle tissue cell ferroptosis degree and the muscle tissue inflammatory injury degree.

Description

Technical Field

[0001] The present invention relates to the technical field of drugs for snake venom bites, and in particular to the application of IMRC-Exo in the preparation of a drug for alleviating local muscle injury of limbs after being bitten by Deinagkistrodon acutus, and the drug. Background Art

[0002] Deinagkistrodon acutus is one of the endemic venomous snakes in Asia. The limb injury caused by it is the main cause of disability in snake bite patients, but its pathogenesis and intervention strategies still need to be further explored and studied. Research shows that after a Deinagkistrodon acutus bite incident, its toxin usually invades the whole body through the local limb, resulting in local muscle tissue edema, ulceration and even necrosis, as well as abnormal systemic coagulation system and abnormal multi-organ functions.

[0003] At present, early strengthening of local limb hemostasis and systemic detoxification and supportive treatment have become the main treatment strategies for Deinagkistrodon acutus bite patients. However, the treatment measures for local limb injury are still only traditional methods such as incision and drainage, negative pressure drainage, etc., which cannot actively contain the pathophysiological process of limb muscle injury caused by snake venom, so the disability rate of snake bite patients is still high.

[0004] Therefore, exploring effective intervention strategies for limb muscle injury after being bitten by Deinagkistrodon acutus will be an important direction to improve the clinical prognosis of snake bite patients.

[0005] In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] Explanation of the abbreviations involved in this application: MSC: Mesenchymal stem cells; IMRC: Immunity and matrix regulatory cells induced by human embryonic stem cells with unlimited stable amplification and totipotent differentiation ability as seed cells; Exo: Exosomes; IMRC-Exo: Human embryonic stem cells derived immune and matrix regulatory cells-exosome, abbreviated as hESC-IMRC-Exo.

[0007] The object of the present invention is to confirm the application effect of IMRC-Exo in the treatment of local muscle injury of the limb after Agkistrodon acutus bite through research, and on this basis, explore the research and development and clinical transformation of IMRC-Exo exosome drugs; at the same time, research and develop a new and highly effective drug for alleviating local muscle injury of the limb after Agkistrodon acutus bite.

[0008] In order to achieve the above object of the present invention, the following technical solutions are specifically adopted: The present invention provides an application of IMRC-Exo in the preparation of a drug for alleviating local muscle injury of the limb after Agkistrodon acutus bite.

[0009] Further, the IMRC-Exo refers to exosomes derived from immune and stromal regulatory cells formed by the directional induction and differentiation of human embryonic stem cells.

[0010] Furthermore, the cell surface markers CD105, CD73 and CD90 of the IMRC cells are all >95%, and the expression rate of the immunogenicity-related protein HLA-DR of the IMRC cells is <5%, the expression rate of the immunogenicity-related protein HLA-E is >50%, and the expression rate of the immunogenicity-related protein HLA-G is >50%.

[0011] Further, the local muscle injury of the limb after Agkistrodon acutus bite includes limb swelling, pain and dysfunction; specifically, the pathological manifestations of the local muscle injury include a large number of rapid ferroptosis of local muscle tissues, resulting in pathological injuries such as muscle fiber necrosis, inflammatory infiltration and apoptosis in the local muscle.

[0012] Further, the application is to inject a pharmaceutical dose of IMRC-Exo into the muscle tissues around the Agkistrodon acutus bite site.

[0013] Further, the administration method is to subcutaneously inject 1-2 ml of the preparation, and the concentration of the preparation is 5×10 10 IMRC-Exo particles / mL; Preferably, the preparation is obtained by diluting IMRC-Exo particles with physiological saline.

[0014] The present invention provides a drug for alleviating local muscle injury of the limb after Agkistrodon acutus bite, and the drug includes IMRC-Exo and a pharmaceutically acceptable excipient.

[0015] Further, the dosage form of the drug is an injection.

[0016] Further, the injection is a subcutaneous injection, and the unit dose of the subcutaneous injection is 5×10 10 IMRC-Exo particles / mL.

[0017] Preferably, the subcutaneous injection is obtained by diluting IMRC-Exo particles with physiological saline.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides the application of IMRC-Exo in the preparation of a drug for alleviating local muscle injury of the limb after Agkistrodon acutus snakebite. Through research, the present application has confirmed the application effect of IMRC-Exo in the treatment of local muscle injury of the limb after Agkistrodon acutus snakebite. On this basis, the research and development of exosome drugs of IMRC-Exo and clinical transformation are explored, which is of great significance for the research and development of new and efficient drugs for alleviating local muscle injury of the limb after Agkistrodon acutus snakebite.

[0019] The present invention provides a drug for alleviating local muscle injury of the limb after Agkistrodon acutus snakebite, and the active components of the drug include IMRC-Exo and pharmaceutically acceptable excipients. It has been experimentally obtained that IMRC-Exo at a pharmaceutically effective dose can effectively relieve local muscle injury of the limb after Agkistrodon acutus snakebite. Description of the Drawings

[0020] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0021] Figure 1 It shows the basic conditions of the animals in the sham operation group, snake venom group, and SV+IMRC-Exo group before modeling provided in Example 2 of the present invention; Figure 2 It shows the change diagrams of limb circumference and muscle injury markers of the animals in the sham operation group, snake venom group, and SV+IMRC-Exo group after modeling provided in Example 2 of the present invention; Figure 3 It shows the gross pathological injury conditions of the muscle tissues of the animals in the sham operation group, snake venom group, and SV+IMRC-Exo group at 24 hours after modeling provided in Example 2 of the present invention; Figure 4 It shows the apoptosis diagrams of the muscle tissues of the animals in the sham operation group, snake venom group, and SV+IMRC-Exo group at 24 hours after modeling provided in Example 2 of the present invention; Figure 5 It shows the ferroptosis diagrams of the muscle tissues of the animals in the sham operation group, snake venom group, and SV+IMRC-Exo group at 24 hours after modeling provided in Example 2 of the present invention; Figure 6This shows the inflammatory damage of the muscle tissues of animals in the sham operation group, snake venom group, and SV+IMRC-Exo group 24 hours after modeling provided in Example 2 of the present invention. Detailed implementation manners

[0022] The technical solutions 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, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0023] According to one aspect of the present invention, there is provided an application of IMRC-Exo in the preparation of a drug for alleviating local muscle injury of the limb after being bitten by Deinagkistrodon acutus.

[0024] The present invention provides an application of IMRC-Exo in the preparation of a drug for alleviating local muscle injury of the limb after being bitten by Deinagkistrodon acutus. Through research, the application effect of IMRC-Exo in the treatment of local muscle injury of the limb after being bitten by Deinagkistrodon acutus is confirmed. On this basis, the research and development and clinical transformation of the exosome drug of IMRC-Exo are explored, which has important significance for the research and development of new and efficient drugs for alleviating local muscle injury of the limb after being bitten by Deinagkistrodon acutus.

[0025] It should be noted that after a Deinagkistrodon acutus bite incident occurs, its toxin usually invades the whole body through the local limb, resulting in swelling, pain, skin breakage, ulceration or even necrosis of the local muscle tissue, as well as abnormal systemic coagulation system and abnormal multi-organ functions. Existing treatment measures for such local muscle injuries of the limb are still only traditional methods such as incision and drainage, negative pressure drainage, etc., which cannot actively curb the pathophysiological process of limb muscle injury caused by snake venom, so the disability rate of patients bitten by Deinagkistrodon acutus is still high.

[0026] The present invention discovers in the research that there are obvious differences in the pathological manifestations between local muscle injury of the limb after being bitten by Deinagkistrodon acutus and conventional muscle injuries or muscle injuries caused by reasons such as ischemia and heat stroke. A large number of rapid ferroptosis of cells will occur in the local muscle tissue at the bitten site of Deinagkistrodon acutus, resulting in pathological injuries such as muscle fiber necrosis, inflammatory infiltration, and apoptosis in the local muscle.

[0027] As a novel form of cell death related to iron ion-mediated lipid peroxidation, there is no relevant research on inhibiting ferroptosis in cells after Agkistrodon acutus bite and thus alleviating pathological damages such as muscle fiber necrosis, inflammatory infiltration, and apoptosis in local muscles at the present stage. In the study of local muscle injury in the limb after Agkistrodon acutus bite in this application, it is found that IMRC-Exo can significantly inhibit the ferroptosis phenomenon in local muscles of the limb after Agkistrodon acutus bite, and thus can effectively reduce the degree of local muscle injury caused by Agkistrodon acutus bite. Therefore, the above research in this application can provide a new and effective treatment strategy for limb muscle injury after Agkistrodon acutus bite.

[0028] In a preferred embodiment of the present invention, the IMRC-Exo refers to exosomes derived from immune and stromal regulatory cells formed by directional induction and differentiation of human embryonic stem cells.

[0029] In the above preferred embodiment, the IMRC-Exo is secreted by IMRC cells; The cell surface markers CD105, CD73, and CD90 of the IMRC cells are all >95%, and the expression rate of the immunogenicity-related protein HLA-DR of the IMRC cells is <5%, the expression rate of the immunogenicity-related protein HLA-E is >50%, and the expression rate of the immunogenicity-related protein HLA-G is >50%.

[0030] Preferably, the preparation process of the IMRC-Exo is as follows: 1) Use human embryonic stem cells to direct their differentiation into IMRC with a purity >95%. By controlling the cell surface markers CD105, CD73, and CD90 of the IMRC cells to be all >95% and achieving HLA-DR <5% and HLA-E and HLA-G >50% in the expression of immunogenicity-related proteins, standardized and low-immunogenic IMRC is obtained; 2) Apply methods such as tangential flow, ultrafiltration, ultracentrifugation, chromatography, and immunoaffinity to extract Exo derived from IMRC, and then obtain standard-quality IMRC-Exo through processes such as purification, identification, counting, specific membrane protein expression, and morphological observation.

[0031] In a preferred embodiment of the present invention, the local muscle injury in the limb after Agkistrodon acutus bite includes limb swelling, pain, and dysfunction; specifically, the pathological manifestations of the local muscle injury include a large number of rapid ferroptosis in local muscle tissues, resulting in pathological damages such as muscle fiber necrosis, inflammatory infiltration, and apoptosis in local muscles.

[0032] In a preferred embodiment of the present invention, the application is to inject a pharmaceutical dose of IMRC-Exo into the muscle tissues around the bite site of Deinagkistrodon acutus. It has been experimentally obtained in this application that subcutaneous injection of IMRC-Exo at a pharmaceutically effective dose can effectively reduce local muscle damage in the limb after being bitten by Deinagkistrodon acutus.

[0033] In a preferred embodiment of the present invention, the administration method is to subcutaneously inject 1 - 2 ml of the preparation, and the concentration of the preparation is 5×10 10 IMRC-Exo particles / mL; Preferably, the preparation is obtained by diluting IMRC-Exo particles with physiological saline.

[0034] According to one aspect of the present invention, a drug for reducing local muscle damage in the limb after being bitten by Deinagkistrodon acutus, the drug comprises IMRC-Exo and a pharmaceutically acceptable excipient.

[0035] A drug for reducing local muscle damage in the limb after being bitten by Deinagkistrodon acutus provided by the present invention, the active components of the drug comprise IMRC-Exo and a pharmaceutically acceptable excipient. It has been experimentally obtained that IMRC-Exo at a pharmaceutically effective dose can effectively relieve local muscle damage in the limb after being bitten by Deinagkistrodon acutus.

[0036] In a preferred embodiment of the present invention, the dosage form of the drug is an injection.

[0037] In a preferred embodiment of the present invention, the injection is a subcutaneous injection, and the unit dose of the subcutaneous injection is 5×10 10 IMRC-Exo particles / mL.

[0038] As a preferred embodiment, the drug for reducing local muscle damage in the limb after being bitten by Deinagkistrodon acutus is a subcutaneous injection, and the unit dose of the subcutaneous injection is 5×10 10 IMRC-Exo particles / mL.

[0039] The technical solutions of the present invention will be further described below in conjunction with the examples.

[0040] Note: In the following examples of this application, by using an experimental rabbit model, the application effect of IMRC-Exo in the treatment of local muscle damage in the limb after being bitten by Deinagkistrodon acutus was confirmed.

[0041] Example 1 (I). Animal preparation: 1. Before the experiment, the rabbits were fasted for 12 h and allowed free access to water.

[0042] 2. During the experiment, weigh the rabbits, then place the experimental rabbits in an experimental animal fixator, take the prone position, and fix the four limbs. Use a professional hair clipper to perform routine skin preparation on the bilateral lower limbs and right ear of the experimental rabbits, fully expose the left lower limb, and mark the middle segment of the outer side of the left thigh as the injection point for the snake venom, measure and record the thigh circumference at the marked point.

[0043] 3. Use an electrocardiogram monitor to monitor the heart rate and oxygen saturation of the experimental rabbits, and use an ear thermometer to measure its body temperature.

[0044] 4. Select 1 ml / kg of 3% pentobarbital solution and inject it intravenously through the marginal ear vein for anesthesia to complete the animal preparation before model establishment and obtain the experimental rabbits.

[0045] (2). Model establishment: Inject 1.5 mg / kg of Deinagkistrodon acutus snake venom into the experimental rabbits in step (1) through the limb. Observe for 2 h after the injection of the snake venom, and then inject 20 ml of 80 U / kg of anti-Deinagkistrodon acutus snake venom serum intravenously to establish an animal model of Deinagkistrodon acutus snake bite.

[0046] Among them: Select the marked position in the middle segment of the outer side of the left thigh as the injection point for the snake venom injection. Insert the needle vertically to a depth of 5 mm, inject 1.5 mg / kg of the snake venom, and use a cotton swab to press the injection site for 1 min after completion to prevent liquid leakage.

[0047] The snake venom is prepared from Deinagkistrodon acutus snake venom freeze-dried powder. The specific preparation method is: dissolve the Deinagkistrodon acutus snake venom freeze-dried powder in physiological saline to prepare a snake venom with a concentration of 10 mg / ml.

[0048] Example 2 (1). Random grouping and intervention of animals: Experimental grouping: Eighteen New Zealand white rabbits, weighing 2.5 - 3.0 kg, are randomly divided into a sham operation group (Group S), a snake venom group (Group SV), and an SV + IMRC-Exo group (IMRC-Exo treatment group), with 6 rabbits in each group.

[0049] Intervention measures: 1) Group S (sham operation group): Only complete the animal preparation work, do not establish a snake bite model, and inject the same amount of physiological saline as the "SV + IMRC-Exo group" subcutaneously as the other groups.

[0050] 2) Group SV (snake venom group): Complete the animal preparation and establish a snake bite model, and inject the same amount of physiological saline as the "SV + IMRC-Exo group" subcutaneously as the other groups.

[0051] 3) SV + IMRC-Exo group (IMRC-Exo treatment group): Animal preparation and snakebite model establishment were completed, and 7.5×10 10 IMRC-Exo particles diluted with normal saline were subcutaneously injected 2 h after snake venom injection.

[0052] Specifically: IMRC-Exo was prepared with normal saline into a solution of 5×10 10 IMRC-Exo particles / mL with a total volume of 1.5 ml for standby. With the injection point of snake venom as the center, 4 equally spaced positions were selected on a circle with a radius of 0.5 cm, and 6 equally spaced positions were selected on a circle with a radius of 1 cm. A total of 10 injections of IMRC-Exo were made. During injection, the needle was inserted vertically 5 mm, and 0.15 ml of IMRC-Exo preparation (obtained by diluting IMRC-Exo particles with normal saline) was injected.

[0053] (II) Observation indicators: 1. Before model establishment, the general vital signs of experimental animals such as body weight, heart rate, oxygen saturation, and body temperature were recorded.

[0054] 2. At 6 h, 12 h, and 24 h before and after model establishment, the changes in the thigh circumference at the snake venom injection site were regularly measured. At the same time, 2 ml of venous blood samples were drawn, centrifuged to obtain plasma and frozen. The serum levels of muscle injury markers such as creatine kinase (CK) and myoglobin (Mb) were detected by enzyme-linked immunosorbent assay at a later time.

[0055] 3. At 24 h after model establishment, the animals were euthanized, and the surrounding muscle tissue specimens at the snake venom injection point were quickly obtained. Pathological samples were made through steps such as fixation, embedding, and sectioning. At a later time, hematoxylin-eosin staining was used to observe the gross pathological changes of muscle tissue, in situ end labeling was used to detect the apoptosis of muscle tissue, and Prussian blue staining was used to detect the iron deposition level in muscle tissue.

[0056] 4. After sacrificing the animals as above, fresh muscle tissue specimens were obtained, quickly frozen in liquid nitrogen, and then cut into 5-μm frozen sections. The sections were incubated with a kit, and the fluorescence intensity of reactive oxygen species (ROS) in muscle tissue was observed by photographing with a fluorescence microscope.

[0057] In addition, fresh muscle tissue specimens were obtained, rapidly frozen with liquid nitrogen and stored in a -80°C refrigerator for a long time. Samples were taken at a later time for homogenization. Western blotting was used to detect the protein expression level of the apoptosis marker protein cleaved caspase 3, biochemical methods were used to detect the contents of malondialdehyde (MDA) and reduced glutathione (GSH), the activity of superoxide dismutase (SOD), and enzyme-linked immunosorbent assay was used to detect the content of the peroxidant 4-hydroxy-2-nonenal (4-HNE), and the contents of the pro-inflammatory factors interleukin-1β (IL-1β) and interleukin-18 (IL-18) in the muscle tissue.

[0058] (III). Research results: (1). Basic conditions of animals in each group: Before model establishment, the basic vital signs of animals in each group, such as body weight, heart rate, oxygen saturation, body temperature, etc., were all within the normal range, and there were no statistically significant differences among groups (all P>0.05). For details, see Figure 1 .

[0059] Figure 1 This is the basic condition of animals in each group before model establishment provided in this example.

[0060] Figure 1 In Figure 1 , A is the basic condition of the body weight of animals in each group before model establishment; B is the basic condition of the heart rate of animals in each group before model establishment; C is the basic condition of the oxygen saturation of animals in each group before model establishment; D is the basic condition of the body temperature of animals in each group before model establishment. Refer to the random grouping of animals in step (I).

[0061] (2). Changes in limb circumference and muscle injury markers of animals in each group: Figure 2 This is the change diagram of limb circumference and muscle injury markers of animals in each group after model establishment provided in this example. Among them: Figure 2 In Figure 2 , A is the change diagram of limb circumference of animals in each group after model establishment; Figure 2 In , B is the change diagram of the muscle injury marker CK (creatine kinase) of animals in each group after model establishment; Figure 2 In

[0062] In addition, Figure 2It can be seen that before modeling, there were no statistically significant differences in the limb circumference and serum levels of muscle injury markers among the three groups of animals (all P>0.05). After modeling, compared with the S group, the limb circumference and serum levels of muscle injury markers in the SV group and the SV+IMRC-Exo group of animals were significantly increased 6 h after snake venom injection (all P<0.05), suggesting that rabbit limb muscle injury occurred after snake venom injection.

[0063] However, the limb circumference and serum levels of muscle injury markers in the SV+IMRC-Exo group of animals were continuously lower than those in the SV group. Among them, CK was significantly decreased at all time points after modeling, and limb circumference and Mb were significantly decreased at 12 h and 24 h after modeling (all P<0.05), suggesting that IMRC-Exo can reduce the degree of rabbit limb muscle injury caused by snake venom.

[0064] (3)Analysis of the gross pathological damage of muscle tissue in each group of animals after modeling Figure 3 This is the gross pathological damage of muscle tissue in each group of animals at 24 h after modeling provided in this example. Figure 3 In which, S is the sham operation group; SV is the snake venom group; the SV+IMRC-Exo group is the IMRC-Exo treatment group.

[0065] See Figure 3 It can be seen that at 24 h after modeling, the gross pathological analysis of muscle tissue showed that the muscle tissue of animals in the S group was generally normal, while obvious pathological damage such as muscle fiber necrosis and inflammatory infiltration occurred in the muscle tissue of animals in the SV group and the SV+IMRC-Exo group after modeling. However, the degree of gross pathological damage of muscle tissue in the SV+IMRC-Exo group was significantly lighter than that in the SV group.

[0066] (4)Analysis of apoptosis of muscle tissue cells in each group of animals after modeling Figure 4 This is the apoptosis situation diagram of muscle tissue cells in each group of animals at 24 h after modeling provided in this example. Among them: Figure 4 In A, it is a representative picture of detecting apoptosis of muscle tissue cells by in situ end labeling method; Figure 4 In B, it is the AI (apoptosis index) diagram of muscle tissue of each group of animals; Figure 4 In C and D, they are representative protein bands of cleaved caspase 3 and their relative expression level diagrams. In addition, Figure 4 In which, S is the sham operation group; SV is the snake venom group; the SV+IMRC-Exo group is the IMRC-Exo treatment group. Compared with the S group, *P<0.05; compared with the SV group, #P<0.05.

[0067] See Figure 4It was found that at 24 h after modeling, compared with the S group, muscle tissue cell apoptosis occurred in the animals of the SV group and the SV+IMRC-Exo group, manifested as a significant increase in AI, and at the same time, the protein expression level of the apoptosis marker protein cleaved caspase 3 was significantly up-regulated, and the differences between groups were statistically significant (all P<0.05). However, compared with the SV group, the AI and the protein expression level of cleaved caspase 3 in the muscle tissue of the IMRC-Exo group were significantly decreased at 24 h after modeling, and the differences between groups were statistically significant (all P<0.05).

[0068] (V). Analysis of ferroptosis of muscle tissue cells in each group of animals after modeling Figure 5 This is the diagram of ferroptosis of muscle tissue cells in each group of animals at 24 h after modeling provided in this example. Among them: Figure 5 A in it is a representative picture of detecting the iron deposition level in muscle tissue by Prussian blue staining method; Figure 5 B in it is the analysis diagram of the iron deposition level of muscle tissue in each group of animals; Figure 5 C in it is a representative picture of detecting reactive oxygen species (ROS) in muscle tissue by immunofluorescence staining method; Figure 5 D in it is the analysis diagram of the ROS fluorescence intensity of muscle tissue in each group of animals; Figure 5 E in it is the analysis diagram of MDA (malondialdehyde) of muscle tissue in each group of animals; Figure 5 F in it is the analysis diagram of 4-HNE (4-hydroxy-2-nonenal) of muscle tissue in each group of animals; Figure 5 G in it is the analysis diagram of GSH (reduced glutathione) of muscle tissue in each group of animals; Figure 5 H in it is the analysis diagram of SOD (superoxide dismutase) of muscle tissue in each group of animals.

[0069] In addition, Figure 5 S in it is the sham operation group; SV is the snake venom group; the SV+IMRC-Exo group is the IMRC-Exo treatment group. Compared with the S group, *P<0.05; compared with the SV group, #P<0.05.

[0070] See Figure 5 It was found that at 24 h after modeling, compared with the S group, ferroptosis of muscle tissue cells occurred in the animals of the SV group and the SV+IMRC-Exo group, manifested as significant increases in the levels of iron deposition, the fluorescence intensity of ROS, the contents of MDA and 4-HNE, and at the same time, the content of GSH and the activity of SOD were significantly decreased, and the differences between groups were statistically significant (all P<0.05).

[0071] However, compared with the SV group, the levels of iron deposition, ROS fluorescence intensity, MDA and 4-HNE content in muscle tissues of the IMRC-Exo group were significantly decreased at 24 h after modeling. Meanwhile, the content of GSH and the activity of SOD were significantly increased, and the differences between groups were statistically significant (all P<0.05).

[0072] (6) Analysis of the inflammatory injury of muscle tissues in each group of animals after modeling Figure 6 This is the situation of the inflammatory injury of muscle tissues in each group of animals at 24 h after modeling provided in this example. Among them: Figure 6 In A is the diagram of the pro-inflammatory factor IL-1β (interleukin-1β); Figure 6 In B is the diagram of the pro-inflammatory factor IL-18 (interleukin-18). In addition, Figure 6 In S is the sham operation group; SV is the snake venom group; SV+IMRC-Exo group is the IMRC-Exo treatment group. Compared with the S group, *P<0.05; compared with the SV group, #P<0.05.

[0073] See Figure 6 It can be seen that at 24 h after modeling, compared with the S group, the animals in the SV group and the SV+IMRC-Exo group showed inflammatory injury of muscle tissues, manifested as a significant increase in the content of the pro-inflammatory factors IL-1β and IL-18, and the differences between groups were statistically significant (all P<0.05). However, compared with the SV group, the content of IL-1β and IL-18 in muscle tissues of the IMRC-Exo group was significantly decreased at 24 h after modeling, and the differences between groups were statistically significant (all P<0.05).

[0074] 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 them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and 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

1. Application of IMRC-Exo in the preparation of drugs for reducing local muscle damage in limbs after Agkistrodon acutus bites.

2. The use according to claim 1, characterized in that: The IMRC-Exo refers to exosomes derived from immune and matrix regulatory cells formed by directed differentiation of human embryonic stem cells.

3. The use according to claim 2, characterized in that: The IMRC-Exo is secreted by IMRC cells; The cell surface markers CD105, CD73 and CD90 of the IMRC cells are all >95%, and the expression rate of the immunogenicity-related protein HLA-DR of the IMRC cells is <5%, the expression rate of the immunogenicity-related protein HLA-E is >50%, and the expression rate of the immunogenicity-related protein HLA-G is >50%.

4. The use according to claim 1, characterized in that: The local muscle damage of the limbs after the bite of the agkistrodon acutus includes limb swelling, pain and functional impairment; The pathological manifestations of local limb muscle injury include a large number of rapid cell iron death in the local muscle tissue, resulting in pathological damage such as muscle fiber necrosis, inflammatory infiltration, and apoptosis in the local muscle.

5. The use according to claim 1, characterized in that: The application is to inject a pharmaceutical dose of IMRC-Exo into the muscle tissue around the bite site of Agkistrodon acutus.

6. The use according to claim 5, characterized in that: The administration method is to administer 1-2 ml of IMRC-Exo preparation via subcutaneous injection, and the concentration of the IMRC-Exo preparation is 5×10 10 IMRC-Exo particles / mL; The IMRC-Exo preparation is obtained by diluting IMRC-Exo particles with physiological saline.

7. A drug for reducing local muscle damage in limbs after being bitten by Agkistrodon acutus, characterized in that: The drug comprises IMRC-Exo and pharmaceutically acceptable excipients.

8. The drug for alleviating local muscle damage in limbs after being bitten by Agkistrodon acutus according to claim 7, characterized in that: The dosage form of the drug is injection.

9. The drug for alleviating local muscle damage of limbs after Agkistrodon acutus bite according to claim 8, characterized in that: The injection is a subcutaneous injection.

10. The drug for alleviating local muscle damage in limbs after being bitten by Agkistrodon acutus according to claim 9, characterized in that: The unit dose of the subcutaneous injection is 5×10 10 IMRC-Exo particles / mL.

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