Antivenom and its application

Abstract

The present invention discloses an anti-snake venom drug and its application. The drug comprises apolipoprotein E, or can inhibit the hemorrhagic toxicity, myotoxicity, and coagulative toxicity of snake venom by increasing the concentration or expression of apolipoprotein E, and inhibit the activities of snake venom metalloproteinase (SVMP), snake venom phospholipase A2 (PLA2), and snake venom C-type lectin (CTL). The drug has the potential to be used in the medical field as a new broad-spectrum anti-snake venom drug, improving the problem that existing antivenom serum has high specificity and strong limitations.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an anti-snake venom drug and application thereof. Background Art

[0002] Apolipoprotein E (ApoE) is a polymorphic protein that participates in the conversion and metabolism of lipoproteins. Its gene can regulate many biological functions. Currently, three main functions have been discovered: (1) ApoE is a ligand for the LDL receptor and the ligand for the CM remnant receptor of hepatocytes. It is closely related to lipoprotein metabolism; (2) ApoE is polymorphic, and polymorphism is closely related to the determination of individual blood lipid levels and the occurrence and development of atherosclerosis; (3) It participates in the activation of enzymes that hydrolyze fat, participates in immune regulation and the regeneration of neural tissue.

[0003] Snake venom is primarily categorized as hematotoxins, neurotoxins, mixed toxins, and cytotoxins. Hematotoxins, including those from vipers, pit vipers, pit vipers, and five-step snakes, cause rapid swelling, hardening, profuse bleeding, severe pain, and a purple-black discoloration of the skin. Skin necrosis and lymphadenopathy often occur. Neurotoxins, including those from Bungarus bungarus and Bungarus krait, present with mild local symptoms after the bite, but develop acute systemic symptoms hours later, including muscle twitching, difficulty breathing and swallowing, and often death from respiratory muscle paralysis. Mixed toxins, including those from cobra and king cobra venom, contain neurotoxins and symptoms of neurotoxin poisoning. Systemic symptoms are severe and complex, with a high mortality rate. Cytotoxins, found in sea snake venom, primarily damage voluntary muscles. The bite is painless, with an incubation period of 30 minutes to several hours. Symptoms of poisoning include muscle weakness and ptosis, accompanied by severe damage to the heart and kidneys.

[0004] Antivenins are specific antibodies that neutralize the corresponding snake venom. Traditional antivenoms, based on horse serum antibodies, are complex to prepare, expensive, highly specific, and limited in variety. They also have significant side effects and are difficult to obtain in remote areas, significantly limiting their use. Furthermore, the disability rate after snake bites remains high. Therefore, the development of new, broad-spectrum antivenoms is urgently needed for emergency use. Summary of the Invention

[0005] The present invention aims to provide an anti-snake venom drug and its application, improve the problems of high specificity and limited scope of use of traditional anti-snake venom serum, and provide a broad-spectrum anti-snake venom emergency drug.

[0006] The present invention provides an antivenom drug, which comprises a drug with apolipoprotein E as the only active ingredient.

[0007] Optionally, the mass ratio of the amount of apolipoprotein E to snake venom in the drug is snake venom: apolipoprotein E (2-4):1.

[0008] Optionally, the drug neutralizes snake venom by increasing the concentration of apolipoprotein E.

[0009] Optionally, the drug reduces the damage to the body caused by snake venom by resisting the hemorrhagic toxicity, myotoxicity and coagulative toxicity of snake venom.

[0010] Optionally, the hemorrhagic toxic components of the snake venom include snake venom metalloproteinase (SVMP), snake venom phospholipase A2 (PLA2), and snake venom-C type lectin (CTL).

[0011] Optionally, the anti-myotoxicity includes anti-gastrocnemius muscle inflammation, anti-muscle swelling, and anti-muscle lysis.

[0012] The beneficial effects of the present invention include:

[0013] (1) The ApoE provided by the present invention can serve as a target for snake venom, "neutralize" snake venom toxins, and exert a toxicity-reducing effect. It can bind to the main components of snake venom, such as PLA2, SVMP, and CTL, thereby neutralizing the snake venom and achieving the effects of anti-hemorrhagic toxicity, anti-myotoxicity, and anti-coagulant toxicity of snake venom;

[0014] (2) The drug provided by the present invention, which contains ApoE as the main active ingredient, is a broad-spectrum antivenom drug that neutralizes the toxicity of snake venom and is expected to be used for emergency treatment of snakebite patients and reduce the disability rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results of the egg yolk plate experiment verify the inhibitory effect of ApoE on snake venom PLA2 enzyme;

[0016] Figure 2 To verify the inhibitory effect of ApoE on snake venom hemorrhagic toxicity;

[0017] Figure 3 To verify the inhibitory effect of ApoE on muscle swelling induced by snake venom;

[0018] Figure 4 This study verifies the improving effect of ApoE on inflammation and myofiber rupture and dissolution of the gastrocnemius muscle after injection of snake venom. DETAILED DESCRIPTION

[0019] The present invention will be further described through the following examples with reference to the accompanying drawings.

[0020] An embodiment of the present invention provides an antivenom drug, which includes a drug containing apolipoprotein E as a main active ingredient.

[0021] In some embodiments, the mass ratio of the amount of apolipoprotein E in the drug to the snake venom is (2-4):1.

[0022] In some embodiments, the drug comprises an injection.

[0023] In some embodiments, the drug neutralizes snake venom by increasing the concentration of apolipoprotein E.

[0024] In some embodiments, the drug reduces the damage to the body caused by snake venom by resisting the hemorrhagic toxicity, myotoxicity, and coagulative toxicity of snake venom.

[0025] In some embodiments, the hemorrhagic toxic components of the snake venom include SVMP, PLA2, and CTL.

[0026] In some embodiments, the anti-myotoxicity includes anti-gastrocnemius muscle inflammation, anti-muscle swelling, and anti-muscle lysis.

[0027] The method for detecting ApoE binding targets using mass spectrometry in the embodiments of the present invention refers to the document Advancing untargeted metabolomics using data-independent acquisition mass spectrometry technology.

[0028] Example 1

[0029] Example 1 of the present invention provides a snake venom-apolipoprotein membrane hybridization assay for detecting ApoE binding targets, comprising the following steps:

[0030] S1. Soak the PVDF membrane in formaldehyde for 1 minute to activate it, let it air dry at 25°C, soak the PVDF membrane in 1 mL of 120 μg / mL ApoE solution on a shaker, and incubate the PVDF membrane at 25°C for 2 hours.

[0031] S2. After the incubation is completed, the ApoE solution is recovered and the PVDF membrane is rinsed three times with TBST on a shaker at 25°C, each rinse for 10 minutes;

[0032] S3. After washing, the PVDF membrane was incubated with Agkistrodon acutus venom (DA) at 4°C for 12 h.

[0033] S4. After the incubation is completed, the PVDF membrane is rinsed three times with TBST on a shaker, each rinse for 10 minutes; then, the membrane is eluted with glycine hydrochloride buffer at pH 3.0, and the eluate is collected. The ApoE-bound toxin is detected by mass spectrometry. The results are shown in Table 1.

[0034] Table 1 ApoE binding target detection results

[0035]

[0036] Property detection

[0037] 1. Verification of the inhibitory effect of ApoE on PLA2 enzyme, the main component of snake venom

[0038] D1. Mix 1 part by mass of egg yolk and 3 parts by mass of normal saline to prepare the substrate stock solution; then mix the substrate stock solution with CaCl2 in a mass ratio of 3:2 to prepare the substrate solution;

[0039] D2. Prepare 0.6% agarose using 0.05 mol / L NaAc at a pH of 7.5. After boiling, cool the agarose to 50°C. Add 500 μL of the substrate solution prepared in step D1 to a culture dish, mix well with the agarose, and plate. After the gel solidifies, punch wells.

[0040] D3. Add ApoE and snake venom to the wells made in step D2, mix well, and add to each well. Incubate at 37°C for 8 hours. Measure the area of ​​the agarose transparent circle and calculate the inhibition rate to determine the neutralization effect of ApoE on the PLA2 enzyme in the snake venom.

[0041] Different component mixtures were added to each well. Well a was added with 0.9% saline as a blank control; well b was added with 10 μg of snake venom; well c was added with a mixture of 10 μg of snake venom and 2 μg of ApoE; well d was added with a mixture of 10 μg of snake venom and 4 μg of ApoE; and well e was added with a mixture of 10 μg of snake venom and 8 μg of ApoE. Each well was filled to 90 μL with 0.9% saline. The results of the egg yolk plate were as follows. Figure 1 As shown in A; the average area of ​​each group was quantified by Image-J and the differences between the groups were analyzed by ANOVA (analysis of variance). Figure 1 As shown in B.

[0042] 3. ApoE's inhibitory effect on snake venom hemotoxicity, myotoxicity, and coagulopathy

[0043] (1) ApoE's inhibitory effect on hemorrhagic toxicity

[0044] Twelve Kunming mice of similar weight, aged 6-8 weeks, were randomly divided into four groups, including a, b, c, and d, with 3 mice in each group;

[0045] Each group received different treatments on the subcutaneous tissue of the mice's backs:

[0046] Group a was the blank control group, which was injected with 40 μL of 0.9% saline;

[0047] Group b was the positive control group, injected with 30 μg of snake venom;

[0048] Group c was the low-dose ApoE treatment group, which was injected with a mixture of 30 μg snake venom + 8 μg ApoE;

[0049] Group d: high-dose ApoE-treated group, injected with a mixture of 30 μg snake venom + 16 μg ApoE;

[0050] Take the skin of the mouse back where the poison was injected subcutaneously and observe it directly. Figure 2 As shown in; Figure 2 a, b, c, d in the table correspond to the groups;

[0051] The cumulative optical density values ​​of the bleeding sites in each group were analyzed by Image-J, and the results obtained by ANOVA analysis of variance were as follows: Figure 2 As shown in e;

[0052] See also Figure 2 The subcutaneous bleeding area of ​​mice in the low-dose ApoE treatment group and the high-dose ApoE treatment group was significantly reduced, demonstrating that the ApoE provided by the present invention has the activity of reducing the hemorrhagic toxicity of snake venom.

[0053] (2) Inhibitory effect of ApoE on myotoxicity

[0054] Nine Kunming mice of similar weight, aged 6-8 weeks, were randomly divided into three groups, including a, b, and c, with three mice in each group;

[0055] Each group of mice underwent snake venom injection experiments on the right gastrocnemius muscle, and the volume of each group was made up to 100 μL with 0.9% saline;

[0056] Group a was the blank control group, injected with 100 μL of 0.9% saline;

[0057] Group b was the venom injection group, which was injected with 80 μg of snake venom;

[0058] Group c was the experimental group, injected with a mixture of 80 μg snake venom + 30 μg ApoE;

[0059] A mouse gastrocnemius muscle injection model was established to detect the anti-myotoxic activity of ApoE;

[0060] The gastrocnemius muscles of mice in different groups were directly observed. Figure 3 As shown; Figure 3 a, b, and c in the figure correspond to the groups. Grahpad-prism was used to analyze the difference of the average width of the gastrocnemius muscles in each group. The results are as follows: Figure 2 As shown in d.

[0061] The gastrocnemius muscles of mice were taken for H&E staining and Masson staining. The staining results are shown in Figure 4 As shown; Figure 4a, b, and c in the table correspond to the groups;

[0062] See also Figure 3 Compared with group b, ApoE treatment in group c can reduce the muscle swelling caused by snake venom, and the muscle morphology after reduction is close to that in group a;

[0063] See also Figure 4 , Figure 4 The white arrows in the figure indicate neutrophils, and the yellow arrows indicate red blood cells. Compared with group b, ApoE treatment in group c effectively inhibited the dissolution and rupture of gastrocnemius muscle fibers in model mice, and the release of inflammatory cells and bleeding were improved after ApoE treatment.

[0064] (3) ApoE's inhibitory effect on coagulopathy

[0065] Plasma was collected from mice injected with poison into the gastrocnemius muscle 24 hours later and the coagulation function was tested using a coagulometer. The coagulation function was indicated by detecting indicators such as activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen (FIB), and prothrombin international normalized ratio (INR). The results are shown in Table 2:

[0066] Table 2 Inhibitory effect of ApoE on coagulopathy

[0067]

[0068] As shown in Table 2, the coagulation time APTT, PT, and TT of the mice in the venom injection group were significantly prolonged, and the INR was significantly prolonged, exceeding the upper limit of the coagulometer detection. The FIB was significantly depleted, below the lower limit of detection, indicating that the snake venom has strong coagulant toxicity and the coagulation function of the mice in the venom injection group was significantly impaired. The coagulation function of the mice in the ApoE injection group was significantly improved, and all indicators were close to normal values.

[0069] Therefore, the ApoE provided by the present invention has multiple activities such as neutralizing the myotoxicity, inflammation, hemorrhagic toxicity, and coagulative toxicity of snake venom.

[0070] While the embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations of these embodiments are possible. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as set forth in the claims. Furthermore, the invention described herein is susceptible to other embodiments and may be practiced or implemented in a variety of ways.

Claims

1. Use of apolipoprotein E in the preparation of antivenom drugs, characterized in that: The drug has anti-snake venom hemorrhagic toxicity, anti-snake venom myotoxicity and anti-snake venom coagulative toxicity; the toxic components of the snake venom are snake venom metalloproteinase and snake venom-C type lectin; the mass ratio of apolipoprotein E in the drug to snake venom is 1: (2-4).

2. The use according to claim 1, characterized in that The drug directly neutralizes snake venom.

3. The use according to claim 1, characterized in that The anti-snake venom myotoxicity is anti-gastrocnemius muscle inflammation, anti-muscle swelling and anti-muscle lysis.

Images