Application of botulinum toxin type A in the preparation of drugs for alleviating local muscle damage in limbs after bites by Agkistrodon acutus and drugs

Abstract

The present invention provides an application of botulinum toxin type A in the preparation of a drug for reducing local muscle damage to limbs after bites by Agkistrodon acutus, and relates to the technical field of snake venom bite drugs. This application confirms the application effect of botulinum toxin type A in the treatment of local muscle damage to limbs after bites by Agkistrodon acutus through research, and on this basis explores the research and development and clinical transformation of botulinum toxin type A drugs. Specifically, this application is obtained by applying a botulinum toxin type A preparation to the biting muscle tissue of an animal model of Agkistrodon acutus bite for four weeks. Compared with the animal model of Agkistrodon acutus bite, the botulinum toxin type A can effectively reduce the circumference of the animal limbs and the level of muscle damage markers in the serum after treatment, and in the analysis of the degree of pathological damage, the degree of apoptosis of muscle tissue cells, and the degree of inflammatory damage to muscle tissue, the effect improvement is statistically significant.

Description

Technical Field

[0001] The present invention relates to the technical field of snake venom bite medicines, and in particular to an application of botulinum toxin type A in the preparation of a medicine for alleviating local muscle damage in limbs after agkistrodon acutus bites and the medicine. Background Art

[0002] The pit viper is one of the common and highly venomous snake species in my country. Its venom is hemotoxic and cytotoxic, and is usually released into the blood through limb bites, causing local limb swelling and muscle tissue necrosis, seriously affecting limb function and significantly reducing the patient's quality of life.

[0003] Currently, treatments for localized limb muscle injuries caused by Agkistrodon acutus bites primarily include local debridement combined with negative pressure drainage, hyperbaric oxygen therapy, and topical application of traditional Chinese medicine. However, these interventions, often performed after the injury has occurred, have limited therapeutic efficacy, often leading to slow wound healing and even limb infection and dysfunction.

[0004] Therefore, it is necessary and urgent to research and develop a preparation and medicine for treating local muscle injury of limbs caused by bites of Agkistrodon acutus.

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

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

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0008] The present invention provides a use of botulinum toxin type A in the preparation of a drug for alleviating local muscle damage in limbs caused by Agkistrodon acutus bites.

[0009] Furthermore, the local muscle damage of the limbs after the bite of the pit viper includes limb swelling, pain and functional impairment, as well as rapid macrophage M1 polarization and intensified inflammatory response in the local muscle tissue at the pathological level, resulting in pathological damage such as local muscle tissue ulceration, muscle fiber necrosis and apoptosis.

[0010] Furthermore, the application is to inject a pharmaceutical dose of botulinum toxin type A into the muscle tissue around the bite site of Agkistrodon acutus.

[0011] 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.

[0012] Furthermore, the method of injection administration is subcutaneous injection.

[0013] Furthermore, the subcutaneous injection is to subcutaneously inject 0.1 ml of 2 U / ml BTX-A preparation at the 0, 3, 6 and 9 o'clock positions 0.5 cm away from the bite site of the Agkistrodon acutus.

[0014] The present invention provides a medicine for alleviating local muscle damage in limbs after being bitten by agkistrodon acutus. The medicine comprises botulinum toxin type A and pharmaceutically acceptable excipients.

[0015] Furthermore, the dosage form of the drug includes at least one of an injection, an injection, and a lyophilized preparation.

[0016] Furthermore, the drug is a subcutaneous injection;

[0017] Furthermore, 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.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a use of botulinum toxin type A in the preparation of a drug for alleviating localized muscle damage to the limbs after bites by Agkistrodon acutus. This application, through research, confirms the efficacy of botulinum toxin type A in the treatment of localized muscle damage to the limbs after bites by Agkistrodon acutus. Based on this, the application explores the drug development and clinical application of botulinum toxin type A. This is of great significance for the research and development of new and highly effective drugs for alleviating localized muscle damage to the limbs after bites by Agkistrodon acutus.

[0020] The present invention provides a drug for alleviating localized muscle damage in a limb after a bite by agkistrodon acutus. The drug's active ingredients include botulinum toxin type A and pharmaceutically acceptable excipients. Experiments have shown that a pharmaceutically effective dose of botulinum toxin type A can effectively alleviate localized muscle damage in a limb after a bite by agkistrodon acutus. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 This is a diagram showing the basic conditions of the animals in the sham operation group, snake venom group, and SV+BTX-A group before modeling provided in Example 2 of the present invention;

[0023] Figure 2 This is a graph showing changes in limb circumference and muscle injury markers in animals in the sham operation group, snake venom group, and SV+BTX-A group after modeling provided in Example 2 of the present invention;

[0024] Figure 3 This is a diagram showing the gross pathological damage of muscle tissue in the sham-operated group, snake venom group, and SV+BTX-A group 24 hours after modeling provided in Example 2 of the present invention;

[0025] Figure 4 This is a graph showing the cell apoptosis in muscle tissue of animals in the sham operation group, snake venom group, and SV+BTX-A group 24 hours after modeling provided in Example 2 of the present invention;

[0026] Figure 5 This is a diagram showing the inflammatory damage in muscle tissue of animals in the sham operation group, snake venom group, and SV+BTX-A group 24 hours after modeling provided in Example 2 of the present invention;

[0027] Figure 6 This is a graph showing the effect of BTX-A provided in Example 2 of the present invention on the expression of CD68, CD86, and CD206 in rabbit muscle tissue after injection of Agkistrodon acutus venom;

[0028] Figure 7 This is a graph showing the effect of BTX-A on the expression of iNOS and Arg1 in rabbit muscle tissue after injection of Agkistrodon acutus venom provided in Example 2 of the present invention;

[0029] above Figure 2 and Figures 4-7 “*” 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). DETAILED DESCRIPTION

[0030] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0031] First, it should be noted that after a bite by a pit viper, its toxins typically penetrate the body through the local limbs, causing swelling, pain, skin damage, ulceration, and even necrosis of local muscle tissue, as well as systemic coagulation system abnormalities and multiple organ dysfunction. Therefore, the local muscle damage caused by a bite by a pit viper is significantly different from existing local muscle damage to the limbs caused by burns, diabetic ulcers, and other causes. The inventors' research has shown that a bite by a pit viper can lead to rapid macrophage polarization and an intensified inflammatory response in the local muscle tissue, causing ulceration and necrosis of the local muscle tissue, necessitating timely treatment.

[0032] However, existing treatments for this type of localized limb muscle injury still rely solely on traditional methods such as incision and vacuum drainage, which fail to effectively curb the pathophysiological progression of limb muscle damage caused by snake venom. Consequently, the disability rate among patients bitten by Agkistrodon acutus remains high. There are also no effective medications available to treat the rapid ulceration and necrosis of localized muscle tissue caused by Agkistrodon acutus bites.

[0033] Botulinum toxin type A (BTX-A), an exotoxin produced by Clostridium botulinum, has been shown to have a role in reducing inflammatory cell infiltration and inflammatory responses. However, whether BTX-A can mitigate local muscle damage caused by Agkistrodon acutus bites and significantly inhibit the rapid macrophage polarization and exacerbated inflammatory response in local muscle tissue, leading to local muscle ulceration, necrosis, and apoptosis, remains unclear and no relevant research has been reported.

[0034] This application uses the venom of the Agkistrodon acutus to establish a rabbit limb local muscle injury model, explores the potential therapeutic effect of BTX-A on this type of limb local muscle injury, and confirms its therapeutic effect, thereby providing a new treatment strategy for limb local muscle injury caused by Agkistrodon acutus bites.

[0035] According to one aspect of the present invention, a botulinum toxin type A is used in the preparation of a drug for alleviating local muscle damage in limbs caused by Agkistrodon acutus bites.

[0036] The present invention provides a use of botulinum toxin type A in the preparation of a drug for alleviating localized muscle damage to the limbs after bites by Agkistrodon acutus. This application, through research, confirms the efficacy of botulinum toxin type A in the treatment of localized muscle damage to the limbs after bites by Agkistrodon acutus. Based on this, the application explores the drug development and clinical application of botulinum toxin type A. This is of great significance for the research and development of new and highly effective drugs for alleviating localized muscle damage to the limbs after bites by Agkistrodon acutus.

[0037] In a preferred embodiment of the present invention, the local muscle damage of the limbs after the bite of the pit viper includes limb swelling, pain and functional impairment, as well as rapid macrophage M1 polarization and intensified inflammatory response in the local muscle tissue at the pathological level, resulting in pathological damage such as local muscle tissue ulceration, muscle fiber necrosis and apoptosis.

[0038] In a preferred embodiment of the present invention, the application is to inject a pharmaceutical dose of botulinum toxin type A into the muscle tissue around the bite site of Agkistrodon acutus.

[0039] As a preferred embodiment, the present application has found through experiments that subcutaneous injection of a botulinum toxin type A preparation at a pharmaceutically effective dose can effectively reduce local muscle damage in the limbs after being bitten by agkistrodon acutus.

[0040] 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 2 U / ml.

[0041] In a preferred embodiment of the present invention, the method of injection administration is subcutaneous injection.

[0042] In the above preferred embodiment, the subcutaneous injection is to inject 0.1 ml of 2 U / ml BTX-A preparation into the 0, 3, 6 and 9 o'clock positions 0.5 cm away from the bite site of the Agkistrodon acutus.

[0043] As a preferred embodiment, the method of "subcutaneously injecting 0.1 ml of a 2 U / ml BTX-A preparation at 0, 3, 6, and 9 o'clock, 0.5 cm from the center of the bite site of the agkistrodon acutus" has been proven to effectively reduce local limb muscle damage after agkistrodon acutus bite.

[0044] According to one aspect of the present invention, a drug for alleviating local muscle damage in limbs after a bite by agkistrodon acutus is provided, the drug comprising botulinum toxin type A and a pharmaceutically acceptable excipient.

[0045] The present invention provides a drug for alleviating localized muscle damage in a limb after a bite by agkistrodon acutus. The drug's active ingredients include botulinum toxin type A and pharmaceutically acceptable excipients. Experiments have shown that a pharmaceutically effective dose of botulinum toxin type A can effectively alleviate localized muscle damage in a limb after a bite by agkistrodon acutus.

[0046] In a preferred embodiment of the present invention, the dosage form of the drug includes at least one of an injection, an injection, and a lyophilized preparation.

[0047] In a preferred embodiment of the present invention, the drug is a subcutaneous injection;

[0048] 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.

[0049] As a preferred embodiment, the drug for reducing local muscle damage in limbs after agkistrodon acutus bite 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.

[0050] The technical solution of the present invention will be further described below with reference to embodiments.

[0051] Note: The following examples of this application use experimental rabbits to establish an animal model of local limb muscle injury caused by Agkistrodon acutus bites, and confirm the therapeutic effect of botulinum toxin type A (BTX-A) on this type of wound injury.

[0052] Example 1

[0053] (I) Animal preparation:

[0054] 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 and night alternation, free access to drinking water, regular feeding, and regular disinfection for 1 week.

[0055] 2. The night before the experiment, the rabbits were fasted for 12 h and allowed free access to water.

[0056] 3. On the day of the experiment, the experimental rabbits were fixed in a special experimental animal holder, and the weight, heart rate, oxygen saturation, body temperature and other physiological indicators of the experimental animals were measured using a weight scale, electrocardiogram monitor and ear thermometer.

[0057] 4. During the experiment, a professional shaver was used to perform routine skin preparation on the lower limbs and right ears of the experimental rabbits to fully expose the lower limbs and right ears. At the same time, the snake venom injection point was marked on the middle part of the outer left thigh.

[0058] 5. Use 3% pentobarbital solution and inject 1 ml / kg of the drug through the ear vein for anesthesia. Complete the animal preparation before model establishment and obtain the experimental rabbits.

[0059] (2) Model establishment:

[0060] (1) When making the model, extract 1.5 mg / kg of snake venom from the experimental rabbit according to step (1). Insert the needle vertically into the middle of the outer left thigh of the experimental rabbit to a depth of 5 mm. Press the injection site with a cotton swab for 1 minute to prevent liquid leakage. 2 hours after the snake venom injection, pump 20 ml of 80 U / kg anti-Agkistrodon quinquefasciatus venom serum into the rabbit's ear vein to establish an animal model of Agkistrodon acutus bite.

[0061] Before use, the anti-Agamo snake venom serum was diluted with normal saline to 20 ml, with a concentration of 12 U / ml. A total of approximately 20 ml was pumped in. The snake venom was prepared from lyophilized powder of Agkistrodon acutus venom. The specific preparation method was to dissolve the lyophilized powder in normal saline to a concentration of 10 mg / ml.

[0062] (2) All experimental animals were monitored for 6 hours after the administration of snake venom and then returned to the rabbit cage for observation for 18 hours. At the end of the experiment, the experimental animals were euthanized by intravenous injection of 150 mg / kg sodium pentobarbital.

[0063] Example 2

[0064] (I) Animal randomization and intervention:

[0065] (1) Experimental groups:

[0066] Twenty-two healthy male New Zealand white rabbits weighing 3.0±0.2 kg were randomly divided into a sham operation group (S group, n=6), a snake venom group (SV group, n=8), and a SV+BTX-A group (BTX-A treatment group, n=8) using a random number table.

[0067] (2) Intervention measures:

[0068] 1) Group S (sham operation group): Only animal preparation was completed without establishing the snake bite model. In addition, the same volume of normal saline was injected subcutaneously as in the other groups.

[0069] 2) SV group (snake venom group): The snake bite model was established based on the animal preparation, and the same volume of normal saline was injected subcutaneously as in the other groups.

[0070] 3) SV+BTX-A group (BTX-A treatment group): Animal preparation and snake bite model were established as above. During the modeling period, 0.1 ml of 2 U / ml BTX-A preparation was subcutaneously injected at 0, 3, 6, and 9 o'clock positions, 0.5 cm away from the snake venom injection point.

[0071] The preparation method of BTX-A preparation is as follows: 100 U of BTX-A drug is dissolved in 50 ml of normal saline to prepare a BTX-A preparation with a concentration of 2 U / ml.

[0072] (II) Observation indicators:

[0073] 1. Before modeling, record the experimental animals' body weight, heart rate, oxygen saturation, body temperature and other physiological indicators.

[0074] 2. Before modeling and 6h, 12h and 24h after modeling, regularly measure the changes in the limb circumference of the animals. At the same time, collect 2ml of blood sample through the ear vein, centrifuge and obtain the supernatant, and freeze it in a -80℃ deep freezer. At an optional time, use ELISA to detect the serum concentrations of muscle injury markers such as creatine kinase (CK) and myoglobin (Mb).

[0075] 3. At 24 hours after modeling, all experimental rabbits were euthanized, and the biceps femoris tissue of the left thigh was quickly obtained. Muscle tissue samples around the injection point of the snake venom were further obtained. Some of the samples were fixed with 4% paraformaldehyde for 24 hours, and then embedded in paraffin and sectioned to prepare pathological samples. The gross pathological changes of muscle tissue were observed by hematoxylin-eosin staining at an appropriate time, and the degree of cell apoptosis was observed and the cell apoptosis rate was calculated after treatment with the TUNEL method. The proportion of cells positively stained for leukocyte differentiation antigen 68 (CD68), leukocyte differentiation antigen 86 (CD86), leukocyte differentiation antigen 206 (CD206), inducible nitric oxide synthase (iNOS), and arginase 1 (Arg1) in muscle tissue was observed after treatment with immunohistochemistry.

[0076] 4. As above, muscle tissue samples were obtained after the experimental rabbits were sacrificed. Some fresh samples were frozen in a -80°C deep freezer. The protein expression level of cleaved caspase 3 was detected by Western blot, and the content of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) was detected by ELISA at an appropriate time.

[0077] (III) Research results:

[0078] (1) Baseline status of the three groups of animals before modeling:

[0079] Figure 1 This is a diagram showing the basic conditions of the animals in the sham operation group, snake venom group, and SV+BTX-A group before modeling provided in this example. Figure 1 A is the basic information of the weight of each group of animals before modeling; B is the basic information of the heart rate of each group of animals before modeling; C is the basic information of the oxygen saturation of each group of animals before modeling; D is the basic information of the body temperature of each group of animals before modeling; refer to the random grouping of animals in step (1), Figure 1 S is the sham operation group; SV is the snake venom group; SV+BTX-A group is the BTX-A treatment group.

[0080] Depend on Figure 1 It can be seen that before modeling, the basic vital signs of animals in each group, such as body weight, heart rate, oxygen saturation, and body temperature, were all within the normal range, and there were no statistically significant differences between the groups (all P>0.05).

[0081] (2) Effects of BTX-A on local muscle damage in rabbit limbs caused by Agkistrodon acutus bites:

[0082] Figure 2 This is a graph showing changes in limb circumference and muscle injury markers in the sham-operated group, snake venom group, and SV+BTX-A group after modeling provided in this example. Figure 2 Middle A is the change of limb circumference of animals in each group after modeling; Figure 2 Middle B is the change of CK (creatine kinase), a muscle damage marker in each group of animals after modeling; Figure 2 Figure C shows the changes in muscle damage marker Mb (myoglobin) in each group of animals after modeling. Figure 2 BL is the baseline; S is the sham operation group; SV is the snake venom venom group; and the SV+BTX-A group is the BTX-A treatment group. *P < 0.05 compared with the S group; #P < 0.05 compared with the SV group.

[0083] From the above Figure 2 There were no statistically significant differences in limb circumference and muscle damage marker concentrations among the three groups before modeling (all P>0.05). Compared with the S group, the thigh circumference of the SV group and the SV+BTX-A group increased significantly at all time points after modeling, and the serum concentrations of CK and Mb increased significantly, with statistically significant differences among the groups (all P<0.05).

[0084] However, compared with the SV group, the thigh circumference of the SV+BTX-A group was significantly reduced at all time points after modeling, and the serum concentrations of the above two muscle damage markers were significantly reduced. There were statistical differences between the groups (all P<0.05), suggesting that BTX-A can reduce the degree of limb muscle damage in rabbits caused by snake venom.

[0085] (3) Effects of BTX-A on pathological damage of rabbit limb muscle tissue caused by Agkistrodon acutus bite:

[0086] Figure 3 This is a diagram showing the gross pathological damage to the muscle tissue of animals in the sham operation group, snake venom group, and SV+BTX-A group 24 hours after modeling provided in this example. Figure 3 S is the sham operation group; SV is the snake venom group; SV+BTX-A group is the BTX-A treatment group.

[0087] Depend on Figure 3Gross pathological analysis of muscle tissue revealed no obvious morphological abnormalities in the limb muscles of the S group. However, 24 hours after modeling, the muscles of the SV and SV+BTX-A groups showed significant myofiber edema and necrosis, as well as infiltration of granulocytes and neutrophils. However, compared with the SV group, the severity of these pathological injuries in the muscles of the SV+BTX-A group was significantly reduced 24 hours after modeling.

[0088] (4) Effects of BTX-A on apoptosis of rabbit limb muscle tissue cells induced by Agkistrodon acutus bites:

[0089] Figure 4 This is a graph showing the cell apoptosis in muscle tissue of animals in the sham operation group, snake venom group, and SV+BTX-A group 24 hours after modeling provided in this example. Figure 4 Middle A is a representative picture of muscle tissue cell apoptosis detected by TUNEL method; Figure 4 Middle B is the apoptosis rate of muscle tissue of animals in each group; Figure 4 Figures C and D show the representative protein bands of cleaved caspase-3 and their relative expression levels. Figure 4 S represents the sham-operated group; SV represents the snake venom group; and the SV+BTX-A group represents the BTX-A-treated group. *P<0.05 compared with the S group; #P<0.05 compared with the SV group.

[0090] From the above Figure 4 Results showed that compared with the S group, the apoptosis rate of muscle tissue in the SV group and the SV+BTX-A group increased significantly at 24 hours after animal modeling, and the expression level of the apoptosis-related protein cleaved caspase 3 was significantly upregulated, with statistically significant differences between the groups (all P<0.05). However, compared with the SV group, the application of BTX-A significantly reduced the apoptosis rate of muscle tissue at 24 hours after animal modeling and significantly downregulated the protein expression level of cleaved caspase 3, with statistically significant differences between the groups (all P<0.05).

[0091] (5) Effects of BTX-A on inflammatory damage to rabbit limb muscle tissue caused by Agkistrodon acutus bites:

[0092] Figure 5 This is a diagram showing the inflammatory damage to the muscle tissue of animals in the sham operation group, snake venom group, and SV+BTX-A group 24 hours after modeling provided in this example. Figure 5 A in the middle is a diagram of TNF-α (tumor necrosis factor-a); Figure 5 Middle B is a diagram showing the status of IL-6 (interleukin-6); Figure 5 The C in the middle is the IL-10 (interleukin-10) situation diagram. In addition, Figure 5 S represents the sham-operated group; SV represents the snake venom group; and the SV+BTX-A group represents the BTX-A-treated group. *P<0.05 compared with the S group; #P<0.05 compared with the SV group.

[0093] From the above Figure 5 It can be seen that compared with the S group, the levels of pro-inflammatory factors TNF-α and IL-6 in muscle tissue of the SV group and the SV+BTX-A group were significantly increased 24 hours after animal modeling, and the level of anti-inflammatory factor IL-10 was also significantly increased, with statistical differences between the groups (all P < 0.05). However, compared with the SV group, the application of BTX-A significantly reduced the levels of TNF-α and IL-6 in muscle tissue of the animals 24 hours after animal modeling, and further significantly increased the level of IL-10, with statistical differences between the groups (all P < 0.05).

[0094] (6) BTX-A promotes M2 polarization of macrophages in rabbit muscle tissue after Agkistrodon acutus bite:

[0095] Figure 6 This example shows the effect of BTX-A on the expression of CD68, CD86, and CD206 in rabbit muscle tissue after injection of Agkistrodon acutus venom. Figure 6 Middle A is a representative image of leukocyte differentiation antigen 68 (CD68) in muscle tissue detected by immunohistochemical staining; Figure 6 Middle B is a graph showing the proportion of CD68 positive cells in the muscle tissue of animals in each group; Figure 6 Middle C is a representative image of the detection of leukocyte differentiation antigen 86 (CD86) in muscle tissue by immunohistochemical staining; Figure 6 Middle D is a graph showing the proportion of CD86-positive cells in the muscle tissue of animals in each group; Figure 6 Middle E is a representative image of the detection of leukocyte differentiation antigen 206 (CD206) in muscle tissue by immunohistochemical staining; Figure 6 Middle F is a graph showing the proportion of CD206 positive cells in the muscle tissue of animals in each group. Figure 6 S represents the sham-operated group; SV represents the snake venom group; and the SV+BTX-A group represents the BTX-A-treated group. *P<0.05 compared with the S group; #P<0.05 compared with the SV group.

[0096] Figure 7 This example shows the effect of BTX-A on the expression of iNOS and Arg1 in rabbit muscle tissue after injection of Agkistrodon acutus venom. Figure 7 Middle A is a representative image of inducible nitric oxide synthase (iNOS) in muscle tissue detected by immunohistochemical staining; Figure 7 Middle B is a graph showing the proportion of iNOS-positive cells in the muscle tissue of animals in each group; Figure 7 Middle C is a representative image of arginase 1 (Arg1) in muscle tissue detected by immunohistochemical staining; Figure 7 Middle D is a graph showing the proportion of Arg1-positive cells in the muscle tissue of animals in each group. Figure 7 S represents the sham-operated group; SV represents the snake venom group; and the SV+BTX-A group represents the BTX-A-treated group. *P<0.05 compared with the S group; #P<0.05 compared with the SV group.

[0097] From the above Figure 6 、 Figure 7 It can be seen that 24 hours after modeling, macrophage activation was observed in the muscle tissues of animals in the SV group and SV+BTX-A group.

[0098] Compared with the S group, the proportion of cells positive for macrophage surface markers CD68, CD86 and CD206 in the muscle tissue of the SV group and SV+BTX-A group was significantly increased, and the proportion of cells positive for its key proteins iNOS and Arg1 was also significantly increased.

[0099] However, compared with the SV group, the SV+BTX-A group showed that BTX-A significantly reduced the proportion of CD86 and iNOS-positive cells, while further increasing the proportion of CD206 and Arg1-positive cells. These results suggest that BTX-A can enhance the M2 polarization of macrophages, thereby promoting the production of the anti-inflammatory factor IL-10 and reducing the levels of pro-inflammatory factors TNF-α and IL-6, thereby alleviating the degree of inflammatory damage in rabbit muscle tissue after Agkistrodon acutus bite.

[0100] It should be noted that BTX-A of the present application can promote the polarization of macrophages from M1 type to M2 type (M2 type is an anti-inflammatory macrophage), thereby increasing the level of anti-inflammatory factors and reducing the content of pro-inflammatory factors, thereby achieving the effect of alleviating inflammatory damage.

[0101] 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 above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to 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 alleviating 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 drug is a botulinum toxin type A preparation; The botulinum toxin type A preparation is prepared by dissolving botulinum toxin type A in physiological saline, and the concentration of the botulinum toxin type A preparation is 2 U / ml.

4. The use according to claim 1, characterized in that The medicine is for subcutaneous injection.

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