Application of botulinum toxin A in preparation of medicine for relieving limb local muscle injury after bite of agkistrodon acutus and medicine

Abstract

The invention provides application of botulinum toxin A in preparation of a medicine for relieving limb local muscle injury after bite of agkistrodon acutus and the medicine, and relates to the technical field of snake venom bite medicines. According to the application, the application effect of the botulinum toxin A in treatment of local muscle injury of limbs bitten by agkistrodon acutus is confirmed through research, and research and development and clinical transformation of the botulinum toxin A medicine are explored on the basis. Specifically, the A-type botulinum toxin 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 A-type botulinum toxin treatment can effectively reduce the peripheral diameter of an animal limb after agkistrodon acutus bite and the muscle injury marker level in serum; and the effect improvement has statistical significance in analysis of the pathological injury degree, the muscle tissue cell apoptosis 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 botulinum toxin type A in the preparation of drugs for alleviating local muscle damage of limbs after being bitten by Deinagkistrodon acutus, and the drugs. Background Art

[0002] Deinagkistrodon acutus is one of the common highly venomous snake species in China. Its venom has hemotoxicity and cytotoxicity, and is usually released into the blood through limb bites, resulting in local limb swelling and muscle tissue necrosis, seriously affecting limb function and significantly reducing the quality of life of patients.

[0003] At present, the treatment methods for local muscle damage of limbs caused by Deinagkistrodon acutus bites mainly include local debridement combined with vacuum sealing drainage, hyperbaric oxygen therapy, and external application of traditional Chinese medicine, etc. However, these measures are mostly intervened after the above-mentioned injuries occur, and their treatment effects are limited, often resulting in slow wound healing, and even causing limb infection and dysfunction.

[0004] Therefore, it is very necessary and urgent to research and develop a preparation and drug for local muscle damage of limbs caused by Deinagkistrodon acutus bites.

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

[0006] The purpose of the present invention is to confirm the application effect of botulinum toxin type A (BTX-A) in the treatment of alleviating local muscle damage of limbs after being bitten by Deinagkistrodon acutus through research, and on this basis, explore the research and development and clinical transformation of botulinum toxin type A drugs; at the same time, research and develop a new and highly effective drug for alleviating local muscle damage of limbs after being bitten by Deinagkistrodon acutus.

[0007] 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 botulinum toxin type A in the preparation of drugs for alleviating local muscle damage of limbs caused by Deinagkistrodon acutus bites.

[0008] Further, the local muscle damage of limbs after being bitten by Deinagkistrodon acutus includes limb swelling and pain and dysfunction, and on the pathological level, there is rapid M1 polarization of macrophages and exacerbation of inflammatory reactions in local muscle tissues, resulting in pathological damage such as local muscle tissue ulceration, muscle fiber necrosis, and apoptosis.

[0009] Further, the application is to inject a pharmaceutical dose of botulinum toxin type A preparation into the muscle tissues around the bitten site of Deinagkistrodon acutus.

[0010] Furthermore, the botulinum toxin type A preparation is mainly prepared by dissolving botulinum toxin type A in physiological saline, and the concentration of the botulinum toxin type A preparation is 2 U / ml.

[0011] Further, the method of injection administration is subcutaneous injection.

[0012] Furthermore, the subcutaneous injection is to subcutaneously inject 0.1 ml of the BTX-A preparation with a concentration of 2 U / ml at the positions of 0 o'clock, 3 o'clock, 6 o'clock and 9 o'clock, which are 0.5 cm away from the bite site of Deinagkistrodon acutus.

[0013] A drug for reducing local muscle injury of limbs after being bitten by Deinagkistrodon acutus provided by the present invention, the drug comprises botulinum toxin type A and pharmaceutically acceptable excipients.

[0014] Further, the dosage form of the drug includes at least one of injection, injectable solution, and lyophilized preparation.

[0015] Further, the drug is a subcutaneous injection; Further, the subcutaneous injection is a botulinum toxin type A preparation, and the unit dose of the botulinum toxin type A preparation is 2 U / ml.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The application of botulinum toxin type A provided by the present invention in the preparation of a drug for reducing local muscle injury of limbs after being bitten by Deinagkistrodon acutus. Through research, the present application confirms the application effect of botulinum toxin type A in the treatment of local muscle injury of limbs after being bitten by Deinagkistrodon acutus, and on this basis, explores the research and development and clinical transformation of botulinum toxin type A drugs, which has important significance for the research and development of new and highly effective drugs for reducing local muscle injury of limbs after being bitten by Deinagkistrodon acutus.

[0017] A drug for reducing local muscle injury of limbs after being bitten by Deinagkistrodon acutus provided by the present invention, the active components of the drug include botulinum toxin type A and pharmaceutically acceptable excipients. It has been experimentally obtained that botulinum toxin type A at a pharmaceutically effective dose can effectively relieve local muscle injury of limbs after being bitten by Deinagkistrodon acutus. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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 the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0019] Figure 1It is the basic situation diagram of the sham operation group, snake venom group, and SV+BTX-A group animals before modeling provided by Embodiment 2 of the present invention; Figure 2 It is the change diagram of limb circumference and muscle injury markers of the sham operation group, snake venom group, and SV+BTX-A group animals after modeling provided by Embodiment 2 of the present invention; Figure 3 It is the general pathological injury situation diagram of the muscle tissue of the sham operation group, snake venom group, and SV+BTX-A group animals 24 hours after modeling provided by Embodiment 2 of the present invention; Figure 4 It is the cell apoptosis situation diagram of the muscle tissue of the sham operation group, snake venom group, and SV+BTX-A group animals 24 hours after modeling provided by Embodiment 2 of the present invention; Figure 5 It is the inflammatory injury situation diagram of the muscle tissue of the sham operation group, snake venom group, and SV+BTX-A group animals 24 hours after modeling provided by Embodiment 2 of the present invention; Figure 6 It is the influence diagram of BTX-A on the expressions of CD68, CD86, and CD206 in the rabbit muscle tissue after injection with Deinagkistrodon acutus venom provided by Embodiment 2 of the present invention; Figure 7 It is the influence diagram of BTX-A on the expressions of iNOS and Arg1 in the rabbit muscle tissue after injection with Deinagkistrodon acutus venom provided by Embodiment 2 of the present invention; The above Figure 2 and Figures 4 - 7 In the above, "*" indicates that there is a significant difference between the treatment group (SV+BTX-A group) and the S group (sham operation group) (p<0.05); "#" indicates that there is a significant difference between the treatment group (SV+BTX-A group) and the SV group (snake venom group) (p<0.05). Specific embodiments

[0020] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. 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.

[0021] First of all, it should be noted that after an event of being bitten by Deinagkistrodon acutus, its toxin usually invades the whole body through the local limb, thus causing swelling, pain, skin breakage, ulceration and even necrosis of the local muscle tissue, as well as abnormal phenomena such as abnormal systemic coagulation system and abnormal functions of multiple organs. Therefore, the local muscle injury caused by being bitten by Deinagkistrodon acutus is significantly different from the local muscle injury of the limb caused by existing reasons such as burns and diabetic ulcers. Through research by the inventor, it is found that after being bitten by Deinagkistrodon acutus, it will lead to rapid macrophage polarization and exacerbation of inflammatory reaction in the local muscle tissue, resulting in ulceration and necrosis of the local muscle tissue, and timely treatment must be given.

[0022] However, the existing treatment measures for such local muscle injuries of the limb are still only traditional methods such as incision and drainage reduction and negative pressure drainage, which cannot actively contain the pathophysiological process of limb muscle injury caused by snake venom, so the disability rate of patients bitten by Deinagkistrodon acutus is still high. There is also no effective drug in the existing technology for the rapid ulceration and necrosis of local muscle tissue caused by being bitten by Deinagkistrodon acutus.

[0023] Botulinum toxin type A (BTX-A), as an exotoxin produced by Clostridium botulinum, has been found to have a certain role in reducing inflammatory cell infiltration and inflammatory reaction. However, whether BTX-A can reduce the local muscle injury of the limb caused by being bitten by Deinagkistrodon acutus, significantly inhibit the rapid macrophage polarization and exacerbation of inflammatory reaction in the local muscle tissue caused by being bitten by Deinagkistrodon acutus, resulting in ulceration, necrosis and apoptosis of the local muscle tissue, is still unclear so far and there is no relevant research report.

[0024] This application uses the venom of Deinagkistrodon acutus to establish a rabbit local muscle injury model of the limb, explores the potential therapeutic effect of BTX-A on such local muscle injuries of the limb, and confirms its application therapeutic effect, so as to provide a new treatment strategy for the local muscle injury of the limb caused by being bitten by Deinagkistrodon acutus.

[0025] According to one aspect of the present invention, there is provided an application of botulinum toxin type A in the preparation of a drug for reducing local muscle injury of the limb caused by being bitten by Deinagkistrodon acutus.

[0026] The application of botulinum toxin type A provided by the present invention in the preparation of a drug for reducing local muscle injury of the limb after being bitten by Deinagkistrodon acutus. This application has confirmed the application effect of botulinum toxin type A in the treatment of local muscle injury of the limb after being bitten by Deinagkistrodon acutus through research, and on this basis, explores the research and development and clinical transformation of botulinum toxin type A drugs, which has important significance for the research and development of new and highly effective drugs for reducing local muscle injury of the limb after being bitten by Deinagkistrodon acutus.

[0027] In a preferred embodiment of the present invention, the local muscle injury of the limb after being bitten by Deinagkistrodon acutus includes limb swelling, pain and dysfunction, and on the pathological level, rapid M1 polarization of macrophages and aggravated inflammatory response occur in the local muscle tissue, resulting in pathological damage such as local muscle tissue ulceration, muscle fiber necrosis and apoptosis.

[0028] In a preferred embodiment of the present invention, the application is to inject a pharmaceutical dose of botulinum toxin type A preparation into the muscle tissues around the bitten site of Deinagkistrodon acutus.

[0029] As a preferred embodiment, it is obtained through experiments in this application that injecting a pharmaceutical effective dose of botulinum toxin type A preparation subcutaneously can effectively reduce the local muscle injury of the limb after being bitten by Deinagkistrodon acutus.

[0030] In the above preferred embodiment, the botulinum toxin type A preparation is mainly prepared by dissolving botulinum toxin type A in physiological saline, and the concentration of the botulinum toxin type A preparation is 2U / ml.

[0031] In a preferred embodiment of the present invention, the method of injection application is subcutaneous injection.

[0032] In the above preferred embodiment, the subcutaneous injection is to subcutaneously inject 0.1 ml of BTX-A preparation with a concentration of 2U / ml at the positions of 0 o'clock, 3 o'clock, 6 o'clock and 9 o'clock, which are 0.5 cm away from the bitten site of Deinagkistrodon acutus as the center.

[0033] As a preferred embodiment, the method of "subcutaneously injecting 0.1 ml of BTX-A preparation with a concentration of 2U / ml at the positions of 0 o'clock, 3 o'clock, 6 o'clock and 9 o'clock, which are 0.5 cm away from the bitten site of Deinagkistrodon acutus as the center". It is verified that it can effectively reduce the local muscle injury of the limb after being bitten by Deinagkistrodon acutus.

[0034] According to one aspect of the present invention, a drug for reducing the local muscle injury of the limb after being bitten by Deinagkistrodon acutus, the drug includes botulinum toxin type A and pharmaceutically acceptable excipients.

[0035] A drug for reducing the local muscle injury of the limb after being bitten by Deinagkistrodon acutus provided by the present invention, the active components of the drug include botulinum toxin type A and pharmaceutically acceptable excipients. It is obtained through experiments that botulinum toxin type A at a pharmaceutical effective dose can effectively relieve the local muscle injury of the limb after being bitten by Deinagkistrodon acutus.

[0036] In a preferred embodiment of the present invention, the dosage form of the drug includes at least one of injection, injectable solution, freeze-dried preparation.

[0037] In a preferred embodiment of the present invention, the drug is a subcutaneous injection; In a preferred embodiment of the present invention, the subcutaneous injection is a botulinum toxin type A preparation, and the unit dose of the botulinum toxin type A preparation is 2 U / ml.

[0038] As a preferred embodiment, the drug for reducing local muscle injury of the limb after being bitten by Deinagkistrodon acutus is a subcutaneous injection, the subcutaneous injection is a botulinum toxin type A preparation, and the unit dose of the botulinum toxin type A preparation is 2 U / ml.

[0039] Hereinafter, the technical solution of the present invention will be further described in conjunction with embodiments.

[0040] Note: In the following embodiments of this application, an animal model of local muscle injury of the limb caused by being bitten by Deinagkistrodon acutus is established by using experimental rabbits, and the therapeutic effect of botulinum toxin type A (BTX-A) on such wound injuries is confirmed.

[0041] Example 1 (I). Animal preparation: 1. Before the experiment, healthy male New Zealand white rabbits were raised under standard conditions of 20-25 °C room temperature, 60%-80% humidity, 12h / 12h day-night alternation, free drinking water, regular feeding, and regular disinfection for 1 week.

[0042] 2. The night before the experiment, the experimental rabbits were fasted for 12 h and allowed free drinking water.

[0043] 3. On the day of the experiment, the experimental rabbits were fixed in a special experimental animal fixator, and physiological indexes such as the body weight, heart rate, oxygen saturation, and body temperature of the experimental animals were measured using a weighing scale, an electrocardiogram monitor, and an ear thermometer respectively.

[0044] 4. During the experiment, a professional hair clipper was used to perform routine skin preparation on the lower limbs and right ear of the experimental rabbits to fully expose the lower limbs and right ear, and at the same time, the injection site of snake venom was marked in the middle of the outer side of the left thigh.

[0045] 5. A 3% pentobarbital solution was applied, and 1 ml / kg of the drug was injected through the marginal ear vein for anesthesia to complete the animal preparation before model establishment, and experimental rabbits were obtained.

[0046] (II). Model establishment: (1). When making the model, snake venom was extracted according to the body weight of the experimental rabbits in step (I) at a dosage of 1.5 mg / kg. The needle was inserted vertically into the middle of the outer side of the left thigh of the experimental rabbits to a depth of 5 mm. After injection, the injection site was pressed with a cotton swab for 1 min to prevent liquid leakage; 2 h after the snake venom injection, 20 ml of anti-Agkistrodon acutus venom serum at 80 U / kg was pumped into the marginal ear vein of the rabbits to establish an animal model of being bitten by Deinagkistrodon acutus.

[0047] Among them: Before use, the anti-Agkistrodon acutus snake venom serum was diluted to 20 ml with physiological saline to a concentration of 12 U / ml, and a total of about 20 ml was pumped in. The snake venom was prepared from the freeze-dried powder of Agkistrodon acutus snake venom. The specific preparation method was: The freeze-dried powder of Agkistrodon acutus snake venom was dissolved in physiological saline to prepare a snake venom with a concentration of 10 mg / ml.

[0048] (2) All experimental animals were monitored for 6 h after administration of snake venom, and then put back into the rabbit cage for observation for 18 h; at the end of the experiment, the experimental animals were euthanized by intravenous injection of sodium pentobarbital at 150 mg / kg.

[0049] Example 2 (I) Random grouping and intervention of animals: (1) Experimental grouping: Twenty-two healthy male New Zealand white rabbits with a body weight of 3.0 ± 0.2 kg were randomly divided into a sham operation group (group S, n = 6), a snake venom group (group SV, n = 8), and an SV + BTX-A group (BTX-A treatment group, n = 8) using a random number table method.

[0050] (2) Intervention measures: 1) Group S (sham operation group): Only the animal preparation work was completed, and the snake bite model was not established. In addition, the same amount of physiological saline was injected subcutaneously as in other groups.

[0051] 2) Group SV (snake venom group): On the basis of animal preparation, a snake bite model was established. In addition, the same amount of physiological saline was injected subcutaneously as in other groups.

[0052] 3) Group SV + BTX-A (BTX-A treatment group): The animal preparation and the establishment of the snake bite model were completed as above, and during the model establishment period, 0.1 ml of a BTX-A preparation with a concentration of 2 U / ml was injected subcutaneously at the 0 o'clock, 3 o'clock, 6 o'clock, and 9 o'clock positions 0.5 cm away from the center of the snake venom injection point.

[0053] The preparation method of the BTX-A preparation was: 100 U of BTX-A drug was dissolved in 50 ml of physiological saline to prepare a BTX-A preparation with a concentration of 2 U / ml.

[0054] (II) Observation indicators: 1. Before model establishment, the physiological indicators such as the body weight, heart rate, oxygen saturation, and body temperature of the experimental animals were recorded.

[0055] 2. Before and at 6 h, 12 h, and 24 h after model establishment, the changes in the limb circumference of the animals were regularly measured. At the same time, 2 ml of blood samples were collected from the marginal ear vein, centrifuged to obtain the supernatant, and stored in a -80 °C deep cryogenic refrigerator for later use. The serum concentrations of muscle injury markers such as creatine kinase (CK) and myoglobin (Mb) were detected by ELISA.

[0056] 3. At 24 h after modeling, all experimental rabbits were euthanized, and the biceps femoris muscle tissue of the left thigh was quickly obtained. Further, muscle tissue samples around the injection site of snake venom were taken. Some were fixed with 4% paraformaldehyde for 24 h, and then pathological samples were made through steps such as paraffin embedding and sectioning. At a selected time, hematoxylin-eosin staining was used for treatment and then photographed to observe the gross pathological changes of the muscle tissue. TUNEL method was used for treatment and then photographed to observe the degree of cell apoptosis and calculate the cell apoptosis rate. In addition, immunohistochemical staining was used for treatment to observe the proportion of cells positively stained with cluster of differentiation 68 (CD68), cluster of differentiation 86 (CD86), cluster of differentiation 206 (CD206), inducible nitric oxide synthase (iNOS), and arginase 1 (Arg1) in the muscle tissue.

[0057] 4. After sacrificing the experimental rabbits as above, muscle tissue samples were obtained. Some fresh samples were stored in a -80 °C deep freezer, and at a selected time, Western blot was used to detect the protein expression level of cleaved caspase 3, and ELISA was used to detect the contents of tumor necrosis factor-a (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10).

[0058] (III). Research results: (1). Baseline status of the three groups of animals before modeling: Figure 1 This is the basic situation diagram of the sham operation group, snake venom group, and SV+BTX-A group of animals before modeling provided in this example. Figure 1 In it, A is the basic situation of the body weight of each group of animals before modeling; B is the basic situation of the heart rate of each group of animals before modeling; C is the basic situation of the oxygen saturation of each group of animals before modeling; D is the basic situation of the body temperature of each group of animals before modeling. Refer to the random grouping of animals in step (I). Figure 1 In it, S is the sham operation group; SV is the snake venom group; the SV+BTX-A group is the BTX-A treatment group.

[0059] As Figure 1 can be seen, the basic vital signs such as body weight, heart rate, oxygen saturation, and body temperature of each group of animals before modeling were all within the normal range, and there were no statistically significant differences among groups (all P>0.05).

[0060] (2). Effect of BTX-A on local muscle injury of rabbit limbs caused by Agkistrodon acutus snake bite: Figure 2 This is the change diagram of limb circumference and muscle injury markers of the sham operation group, snake venom group, and SV+BTX-A group of animals after modeling provided in this example. Among them: Figure 2In Figure A, the changes in limb circumference of animals in each group after modeling are shown; Figure 2 In Figure B, the changes in the muscle injury marker CK (creatine kinase) of animals in each group after modeling are shown; Figure 2 In Figure C, the changes in the muscle injury marker Mb (myoglobin) of animals in each group after modeling are shown. Additionally, Figure 2 In Figure BL is the baseline; S is the sham operation group; SV is the snake venom group; the SV+BTX-A group is the BTX-A treatment group. Compared with the S group, *P<0.05; compared with the SV group, #P<0.05.

[0061] From the above Figure 2 it can be seen that there were no statistically significant differences in limb circumference and muscle injury marker concentrations among the three groups of animals before modeling (all P>0.05). Compared with the S group, the thigh circumferences of the SV group and the SV+BTX-A group were significantly increased at each time point after modeling, and at the same time, the serum concentrations of CK and Mb were significantly increased, and there were statistically significant differences between groups (all P<0.05).

[0062] However, compared with the SV group, the thigh circumferences of the SV+BTX-A group were significantly reduced at each time point after modeling, and at the same time, the serum concentrations of the above two muscle injury markers were significantly decreased, and there were statistically significant differences between groups (all P<0.05), indicating that BTX-A can reduce the degree of limb muscle injury caused by snake venom in rabbits.

[0063] (3) Effects of BTX-A on pathological injury of limb muscle tissue in rabbits bitten by Deinagkistrodon acutus: Figure 3 This is a diagram showing the gross pathological injury of muscle tissues of animals in the sham operation group, snake venom group, and SV+BTX-A group 24 hours after modeling provided in this example. Among them: Figure 3 In Figure, S is the sham operation group; SV is the snake venom group; the SV+BTX-A group is the BTX-A treatment group.

[0064] From Figure 3 the gross pathological analysis of muscle tissues showed that there were no obvious morphological abnormalities in the limb muscle tissues of animals in the S group, but obvious pathological injury manifestations such as myofiber edema, necrosis, and infiltration of medium and fine granulocytes were visible in the muscle tissues of animals in the SV group and the SV+BTX-A group 24 hours after modeling. However, compared with the SV group, the degree of the above pathological injury in the muscle tissues of animals in the SV+BTX-A group 24 hours after modeling was significantly reduced.

[0065] (4) Effects of BTX-A on apoptosis of limb muscle tissue cells in rabbits bitten by Deinagkistrodon acutus: Figure 4 This is a diagram showing the apoptosis of muscle tissues of animals in the sham operation group, snake venom group, and SV+BTX-A group 24 hours 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 the TUNEL method; Figure 4 In B, it is a graph of the apoptosis rate of muscle tissue of animals in each group; Figure 4 In C and D, they are representative protein bands of cleaved caspase-3 and graphs of their relative expression levels. Additionally, Figure 4 In S, it is the sham operation group; in SV, it is the snake venom group; in the SV+BTX-A group, it is the BTX-A treatment group. Compared with the S group, *P<0.05; compared with the SV group, #P<0.05.

[0066] From the above Figure 4 It can be seen that compared with the S group, the apoptosis rate of muscle tissue in the SV group and the SV+BTX-A group significantly increased at 24 h after animal modeling, and at the same time, the expression level of the apoptosis-related protein cleaved caspase 3 was significantly up-regulated, and there were statistical differences in the comparison between groups (both P<0.05). However, compared with the SV group, applying BTX-A could significantly reduce the apoptosis rate of muscle tissue at 24 h after animal modeling, and at the same time, significantly down-regulate the protein expression level of cleaved caspase 3, and there were statistical differences in the comparison between groups (both P<0.05).

[0067] (5) Effects of BTX-A on inflammatory injury of rabbit limb muscle tissue caused by Agkistrodon acutus snake bite: Figure 5 This is a graph of the inflammatory injury of muscle tissue of animals in the sham operation group, snake venom group, and SV+BTX-A group at 24 h after modeling provided in this example. Among them: Figure 5 In A, it is a graph of TNF-α (tumor necrosis factor-α); Figure 5 In B, it is a graph of IL-6 (interleukin-6); Figure 5 In C, it is a graph of IL-10 (interleukin-10). Additionally, Figure 5 In S, it is the sham operation group; in SV, it is the snake venom group; in the SV+BTX-A group, it is the BTX-A treatment group. Compared with the S group, *P<0.05; compared with the SV group, #P<0.05.

[0068] From the above Figure 5 It can be seen that compared with the S group, the contents of pro-inflammatory factors TNF-α and IL-6 in muscle tissue in the SV group and the SV+BTX-A group significantly increased at 24 h after animal modeling, and at the same time, the content of the anti-inflammatory factor IL-10 also significantly increased, and there were statistical differences in the comparison between groups (both P<0.05). However, compared with the SV group, applying BTX-A could significantly reduce the contents of TNF-α and IL-6 in muscle tissue at 24 h after animal modeling, and at the same time, further significantly increase the content of IL-10, and there were statistical differences in the comparison between groups (both P<0.05).

[0069] (6) BTX-A promotes the M2 polarization of macrophages in rabbit muscle tissue after Agkistrodon acutus snakebite: Figure 6 This is the diagram showing the effects of BTX-A provided in this example on the expressions of CD68, CD86, and CD206 in rabbit muscle tissue after injection with Agkistrodon acutus snake venom. Figure 6 In it, A is the representative picture of detecting cluster of differentiation 68 (CD68) in muscle tissue by immunohistochemical staining; Figure 6 In it, B is the diagram of the positive cell ratio of CD68 in the muscle tissue of each group of animals; Figure 6 In it, C is the representative picture of detecting cluster of differentiation 86 (CD86) in muscle tissue by immunohistochemical staining; Figure 6 In it, D is the diagram of the positive cell ratio of CD86 in the muscle tissue of each group of animals; Figure 6 In it, E is the representative picture of detecting cluster of differentiation 206 (CD206) in muscle tissue by immunohistochemical staining; Figure 6 In it, F is the diagram of the positive cell ratio of CD206 in the muscle tissue of each group of animals. Figure 6 In it, S is the sham operation group; SV is the snake venom group; the SV+BTX-A group is the BTX-A treatment group. Compared with the S group, *P<0.05; compared with the SV group, #P<0.05.

[0070] Figure 7 This is the diagram showing the effects of BTX-A provided in this example on the expressions of iNOS and Arg1 in rabbit muscle tissue after injection with Agkistrodon acutus snake venom. Figure 7 In it, A is the representative picture of detecting inducible nitric oxide synthase (iNOS) in muscle tissue by immunohistochemical staining; Figure 7 In it, B is the diagram of the positive cell ratio of iNOS in the muscle tissue of each group of animals; Figure 7 In it, C is the representative picture of detecting arginase 1 (Arg1) in muscle tissue by immunohistochemical staining; Figure 7 In it, D is the diagram of the positive cell ratio of Arg1 in the muscle tissue of each group of animals. Figure 7 In it, S is the sham operation group; SV is the snake venom group; the SV+BTX-A group is the BTX-A treatment group. Compared with the S group, *P<0.05; compared with the SV group, #P<0.05.

[0071] From the above Figure 6 , Figure 7 it can be seen that at 24 h after modeling, macrophage activation can be seen in the muscle tissue of animals in the SV group and the SV+BTX-A group.

[0072] Compared with the S group, the proportion of positive cells stained with surface markers CD68, CD86, and CD206 of macrophages in the muscle tissues of the SV group and the SV+BTX-A group was significantly increased. At the same time, the proportion of positive cells stained with key proteins iNOS and Arg1 was also significantly increased.

[0073] However, compared with the SV group, the application of BTX-A in the SV+BTX-A group could significantly reduce the proportion of positive cells of CD86 and iNOS, and at the same time further increase the proportion of positive cells of CD206 and Arg1. These results suggest that BTX-A can promote the production of anti-inflammatory factor IL-10 and reduce the levels of pro-inflammatory factors TNF-α and IL-6 by enhancing the M2 polarization of macrophages, thereby reducing the degree of inflammatory injury in the rabbit muscle tissues after Agkistrodon acutus snakebite.

[0074] It should be noted that in this application, BTX-A can promote the polarization of macrophages from M1 type to M2 type (M2 type is an anti-inflammatory type of macrophage), thereby increasing the level of anti-inflammatory factors and reducing the content of pro-inflammatory factors, achieving the effect of reducing inflammatory injury.

[0075] 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 botulinum toxin type A in the preparation of drugs for reducing local muscle damage in limbs after bites by Agkistrodon acutus.

2. 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 local muscle damage of the limbs includes the rapid polarization of macrophages M1 and the intensification of inflammatory response in the local muscle tissue, resulting in pathological damage such as local muscle tissue ulceration, muscle fiber necrosis and apoptosis.

3. The use according to claim 1, characterized in that: The application is to inject a pharmaceutical dose of botulinum toxin type A into the surrounding muscle tissue of the bite site of the pit viper.

4. The use according to claim 3, characterized in that: The botulinum toxin type A preparation is mainly prepared by dissolving botulinum toxin type A in physiological saline, and the concentration of the botulinum toxin type A preparation is 2 U / ml.

5. The use according to claim 3, characterized in that: The method of injection administration is subcutaneous injection.

6. The use according to claim 5, characterized in that: The subcutaneous injection is to subcutaneously inject 0.1 ml of BTX-A preparation with a concentration of 2 U / ml at the positions of 0, 3, 6 and 9, 0.5 cm away from the bite site of the Agkistrodon acutus.

7. A drug for reducing local muscle damage in limbs after being bitten by Agkistrodon acutus, characterized in that: The drug comprises botulinum toxin type A 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 includes at least one of an injection, an injection, and a lyophilized preparation.

9. The drug for alleviating local muscle damage of limbs after Agkistrodon acutus bite according to claim 8, characterized in that: The medicine is an injection, and 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 subcutaneous injection is a botulinum toxin type A preparation; The unit dose of the botulinum toxin type A preparation is 2 U / ml.

Images