CN114432297B - Application of Zaragozic acid A in treatment of clostridium perfringens Epsilon toxin poisoning - Google Patents

Abstract

The invention discloses application of Zaragozic acid A in treatment of clostridium perfringens Epsilon toxin poisoning. The invention provides an application of Zaragozic acid A in any one of the following: 1) As an ETX toxin inhibitor; 2) Preventing or treating a disease in an animal caused by an ETX toxin; 3) Treating a disease caused by an ETX toxin; 4) Inhibiting toxicity of ETX toxin; 5) Neutralize ETX toxin. Experiments prove that ZA has good toxic neutralization effect on ETX toxin. At the cellular level, ZA has a good inhibition effect on the toxicity of ETX toxin, and in animal experiments, ZA shows a strong protection effect on animals subjected to ETX toxin challenge. Therefore, ZA is expected to be an important agent and drug for preventing and treating infection caused by ETX toxin.

Description

Application of Zaragozic acid A in treatment of clostridium perfringens Epsilon toxin poisoning

Technical Field

The invention belongs to the technical field of biology, and relates to application of Zaragozic acid A in treatment of clostridium perfringens Epsilon toxin poisoning.

Background

Clostridium perfringens (Clostridium perfringens), old known as Clostridium welchii, produces a variety of exotoxins, such as alpha, beta, gamma, epsilon, theta, delta, eta, iota, kappa, lambda, mu, v, and the like. The 6 major exotoxins produced (α, β, ε, iota, CPE and NetB) can be classified as A, B, C, D, E, F and seven types G. Among them, epsilon toxin (Epsilon toxin, ETX toxin) is produced by Clostridium perfringens type B and D, and causes lethal enterotoxemia in ruminants. The disease is acute in onset, rapid in course of disease and high in death rate, and causes huge loss to animal husbandry. In addition, ETX toxin can also cause multiple sclerosis in humans. The toxicity of ETX toxin was found to be second only to that of botulinum toxin and tetanus toxin, and was listed as a class B bioterrorism agent by the united states centers for disease prevention and control (CDC), a potential threat to human safety.

The animal kidneys and brain are the major target organs for the ETX toxin. When ETX toxin enters the animal body, it can accumulate in the kidney and brain in large quantities, causing congestion and edema, destroying the blood brain barrier, and causing death of the animal. However, there are few cells sensitive to ETX toxin, mainly murine renal cortical collecting duct (mpkcdcl 4) cells, human renal leiomyoma (G-402) cells, fischer murine thyroid (FRT) cells, and human renal granular cell carcinoma (ACHN) cells, with the most sensitive cells being canine renal epithelial (MDCK) cells. When ETX toxin attacks cells, it first binds to specific receptors on the cell surface, forms a heptameric complex on lipid rafts, and forms a large pore on the cell membrane surface, resulting in a change in the permeability of the cell membrane, followed by Na ⁺ Internal flow and K ⁺ Efflux, eventually leading to Ca ²⁺ Influx, eventually causing cell death.

At present, an effective treatment means is lacking in clinical treatment aiming at the ETX toxin poisoning, so that a new ETX toxin poisoning treatment medicine and a new ETX toxin poisoning treatment method are quite necessary to be searched.

Disclosure of Invention

An object of the present invention is to provide use of Zaragozic acid A (hereinafter referred to as ZA).

The invention provides an application of Zaragozic acid A in any one of the following or preparing a product with any one of the following functions:

1) As an ETX toxin inhibitor;

2) Preventing or treating an animal disease caused by ETX toxin;

3) Treating a disease caused by an ETX toxin;

4) Inhibiting toxicity of ETX toxin;

5) Neutralizing the ETX toxin;

6) Reducing the toxic effect of the ETX toxin on cells;

7) Improving the ability of the animal to resist the attack of ETX toxin.

The above-mentioned animal diseases caused by ETX toxin are exemplified in the examples of the present invention by animal toxicities caused by ETX toxin. In an embodiment of the invention, the ETX toxin is derived from clostridium perfringens.

The application of the substance taking Zaragozic acid A as the active ingredient in any one of the following or preparing the product with any one of the following functions is also within the protection scope of the invention:

1) As an ETX toxin inhibitor;

2) Preventing or treating a disease in an animal caused by an ETX toxin;

3) Treating a disease caused by an ETX toxin;

4) Inhibiting toxicity of ETX toxin;

5) Neutralizing the ETX toxin;

6) Reducing the toxic effect of the ETX toxin on cells;

7) Improving the ability of the animal to resist the attack of ETX toxin.

The animal diseases caused by ETX toxin described above are exemplified in the examples of the present invention by animal toxicity caused by ETX toxin.

It is another object of the invention to provide a product.

The active ingredient of the product provided by the invention is Zaragozic acid A.

The product has any one of the following functions:

1) As an ETX toxin inhibitor;

2) Preventing or treating a disease in an animal caused by an ETX toxin;

3) Treating a disease caused by an ETX toxin;

4) Inhibiting toxicity of ETX toxin;

5) Neutralizing the ETX toxin;

6) Reducing the toxic effect of the ETX toxin on cells;

7) Improving the ability of the animal to resist the attack of ETX toxin.

In the examples of the present invention, the cells are exemplified by MDCK cells, and the animals are exemplified by mice.

The invention has two main aspects: one aspect is to verify ZA at the cellular level for its inhibitory effect on ETX toxins. The inventors have demonstrated, based on previous work accumulation, the inhibitory effect of ZA on ETX toxin toxicity in MDCK cells. The results show that: ZA effectively inhibits the toxic effects of ETX toxin on MDCK cells and is concentration dependent. Therefore, the invention shows that ZA has important application prospect in the treatment of ETX toxin poisoning.

Another aspect of the invention is to verify the effect of ZA in curing animal poisoning caused by ETX toxin through animal experiments. ZA is effective in inhibiting toxicity of ETX toxin, increasing survival rate of ETX toxin-poisoned animals, and protecting animals against ETX toxin infection. Therefore, ZA can be used in the preparation of a related medicament or agent for preventing or treating ETX toxin intoxication in an animal.

The ZA molecule related by the invention has clear structure and simple acquisition. The preparation method of the ETX toxin is mature and mainly obtained in a recombinant expression mode. ETX toxin is most sensitive to MDCK cells and is therefore used in the present invention for the study of the rescue of ZA poisoning. The validation method conforms to the category of classical methods in the field of toxin research.

ZA showed an inhibitory effect on ETX toxin toxicity in both MDCK cytotoxicity experiments and mouse challenge experiments, indicating that MDCK cell mortality was reduced and that 100% of mice survived when ZA-treated mice were again challenged with a complete lethal dose of ETX toxin. According to the invention, ZA has a good treatment effect on ETX toxin poisoning, has a good application prospect in animal husbandry, and has a very important significance on biological safety of human society.

The invention has the beneficial effects that:

1. the ZA related to the invention has good neutralizing effect on the toxicity of ETX toxin. At the cellular level, ZA has a good inhibition effect on the toxicity of ETX toxin, and in animal experiments, ZA shows a strong protection effect on animals subjected to ETX toxin challenge. Therefore, ZA is expected to be an important agent and drug for preventing and treating infection caused by ETX toxin.

2. The ZA molecule related to the invention has simple structure, is easy to obtain in the market, has good application prospect in animal husbandry and has very important significance for human social safety.

The inventor has been engaged in ETX toxin-related research for a long time, and found that lipid-lowering drug ZA (Zaragozic acid A, CAS No. 144541-82-2) can destroy lipid raft structure on cell surface, weaken toxic effect of ETX toxin on cells, completely protect mice against attack of lethal dose ETX toxin, and reduce death rate according to toxic effect characteristics.

Drawings

FIG. 1 is a SDS-PAGE result of ETX toxin purification.

FIG. 2 is a molecular structure diagram of ZA.

Figure 3 is a graph of the results of various concentrations of ETX toxin versus MDCK cytotoxicity.

Fig. 4 is a graph showing the results of ZA on inhibiting ETX toxin cytotoxicity on MDCK.

Figure 5 is a graph of survival of mice after challenge with different concentrations of ETX toxin.

Fig. 6 is a graph of survival of mice after ZA challenge to ETX toxin.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The lipid-lowering drug ZA (Zaragozic acid A, CAS number 144541-82-2) in the following examples has a molecular structure as shown in FIG. 2.

Example 1 use of ZA to inhibit ETX toxin

1. Expression and purification of ETX toxin

The recombinant strain BL21 (DE 3) of ETX toxin stored in the laboratory is used for purifying and obtaining ETX toxin with higher purity for subsequent research.

The ETX toxin recombinant strain BL21 (DE 3) is a recombinant strain obtained by introducing a recombinant vector into BL21 (DE 3);

the recombinant vector is obtained by replacing BamHI and XhoI enzyme cutting sites of pGEX-4T-1 vector (Cytiva, catalog number 28954549) with ETX toxin gene (sequence 1), the ETX toxin gene and GST label on the vector are fused and expressed to obtain the ETX toxin fused with the GST label, the amino acid sequence of the ETX toxin fused with the GST label is sequence 2 in the sequence table, wherein the 228 th to 488 th positions of the sequence 2 are ETX toxin, and the 1 st to 226 th positions are GST label.

The specific steps of the expression and purification of the ETX toxin are as follows:

1. culture and induction of ETX toxin recombinant strain BL21 (DE 3)

The stored ETX toxin recombinant strain BL21 (DE 3) was taken out from a refrigerator at-80 ℃ and transferred into 5ml of LB liquid medium containing 100. Mu.g/ml ampicillin (5 g of peptone, 2.5g of agar powder and 5g of NaCl were added to 500ml of the medium, and the balance water) at a ratio of 1 ₆₀₀ The value is between 0.6 and 0.8, IPTG is added to a final concentration of 1mM, and the mixture is cultured at 16 ℃ overnight at 180r/min to obtain a culture solution.

2. Preparation of protein mixture

Centrifuging the culture solution obtained in the step 1 at 4 ℃ at 8000r/min for 15min to collect thallus, discarding the supernatant, resuspending the thallus with PBS, centrifuging at 8000r/min for 15min, discarding the supernatant, adding 80ml of solution A (containing 1L A solution, 8.18g NaCl,0.2g KCl,3.58g Na) ₂ HPO ₄ ，0.25g KH ₂ PO ₄ 0.62g DTT, pH 7.3, and balance water) and then the cells were sonicated (power 75%, 3s burst, 2s stop). Centrifuging the crushed bacteria solution at 12000r/min for 15min at 4 deg.C, and filtering the supernatant with 0.45 μm filter to obtain protein mixture.

3. Purification of ETX toxins using a purifier

(1) Washing a A, B pump of the purifier with sterile water at a flow rate of 3ml/min;

(2) Flushing the B pump with solution B (1L B solution added with 7.88g Tris-HCl,3.07g 10mM reduced glutathione, 0.62g DTT, pH 8.0, and balance water) at flow rate of 3ml/min;

(3) Will GSTrap ^TM HP column (Cytiva, cat: 17528201) was set up above purification, and A pump and GSTrap were flushed with A solution at a flow rate of 3ml/min ^TM An HP chromatographic column;

(4) The protein mixed solution prepared in the step 2 is processed by the GSTrap treated in the step 3 ^TM Loading the HP chromatographic column at the flow rate of 2ml/min;

(5) Flushing of GSTrap with solution A ^TM HP chromatographic column with flow rate of 3ml/min;

(6) Eluting with solution B in gradient mode and GSTrap ^TM The HP column bound protein at a flow rate of 3ml/min and the eluate of the eluted peak was collected and analyzed by SDS-PAGE.

As shown in FIG. 1, the solution A is the product collected in the above step (5), and the solution B is the product collected in the above step (6); penetrating into the product collected in the step (4); it can be seen that, after purification, the GST tag-fused ETX toxin (named GST-ETX) with higher purity and size of 56kDa is obtained, the amino acid sequence is sequence 2, the concentration is 4mg/ml, and the GST tag-fused ETX toxin can be used for subsequent experiments.

The ETX toxins used in the following experiments were GST-ETX.

2. ZA verification of toxicity inhibition ability of ETX toxin to MDCK cells

1. Preparation of 96-well cell plate from MDCK cells

The invention adopts the inhibitory activity of the ETX toxin to cause MDCK cell death as an evaluation standard for verifying the toxic inhibitory effect of ZA on the ETX toxin. MDCK cells (Wuhan Punuo Sai Life technologies, inc., catalog number: CL-0154) were diluted to 10% with 10% (volume percent) FBS-containing DMEM medium before use ⁵ one/mL, spread in 96-well plates, each well containing 10 ⁴ The individual cells were cultured in a cell incubator for 24 hours to prepare a 96-well plate containing MDCK cells.

2. Determination of the half-lethal dose of ETX toxin to MDCK cells

Experimental groups: diluting the one-prepared ETX toxin GST-ETX with DMEM medium containing 10% (volume percentage) FBS to a final concentration of 16000, 8000, 4000, 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.096, 1.95, 0.97, 0.488, 0.244, 0.122ng/ml for 18 gradients, and adding it to the 96-well plate containing MDCK cells of 1 above in a volume of 100 μ l/well;

blank control group: the 96-well plate containing MDCK cells of the above 1 was added with 100. Mu.l/well of DMEM medium containing 10% (volume percentage) FBS as a control group;

after the above groups are incubated in a cell culture box for 1h, the culture medium is discarded, PBS is washed for 3 times, and 100 mul/well of DMEM medium containing 10% (volume percentage content) FBS and 20 mul/well MTS are added (the step of

Aqueous One Solution Cell Proliferation Assay Kit, program, usa under the product number G3581), and after incubation in a Cell culture chamber for 3 hours, the absorbance at 492nm was measured, and the survival rate of MDCK cells was calculated according to the following formula.

Cell viability = absorbance at 492nm of experimental group/absorbance at 492nm of control group

The results are shown in figure 3, and the half lethal dose of ETX calculated to be cytotoxic to MDCK is approximately 100ng/ml.

3. Incubation of varying concentrations of ZA with MDCK cells

Zaragozic acid A (Santa Cruz, USA, cat # sc 302001) was dissolved in 0.6ml DMSO, diluted to 2mM stock solution by adding 2.7ml DMEM medium containing 10% (volume percent) FBS, and then diluted to 8 gradients of 600, 300, 150, 75, 37.5, 18.75, 9.375, 0. Mu.g/ml in DMEM medium containing 10% FBS, and added to the 96-well plate containing MDCK cells of 1 above in a volume of 100. Mu.l/ml; and (3) incubating the cells with MDCK cells for 30min in a cell culture box to obtain a ZA and MDCK cell co-incubation cell plate.

4. ETX toxin attacking cells

Experimental group (100 ng/mL GST-EXT + ZA): diluting the ETX toxin GST-ETX prepared in the previous step to a final concentration of 100ng/ml by using a DMEM medium containing 10% (volume percentage) FBS, and respectively adding ZA and MDCK cell co-incubation cell plates obtained in the previous step 3 according to 100 mu l/pore volume;

control group (ZA 0): the ETX toxin GST-ETX prepared above was diluted to a final concentration of 100ng/ml with DMEM medium containing 10% (volume percentage content) FBS, and the above 96-well plate containing MDCK cells 1 was added in a volume of 100 μ l/well.

After incubation for 1h in the cell incubator, the medium is discarded, PBS is washed for 3 times, 100 mul/well of DMEM medium containing 10% (volume percentage content) FBS and 20 mul/well MTS are added, after incubation for 3h in the cell incubator, the light absorption value at 492nm is detected, and the survival rate is calculated according to the formula in 2.

ZA shows that, as compared with the control group (ZA is 0), the addition of ZA has a significant inhibitory effect on MDCK cytotoxicity caused by ETX toxin, and the addition of ZA at a concentration of 300 to 600 μ g/ml effectively reduces the mortality of MDCK cells caused by ETX toxin, as shown in fig. 4.

3. ZA protective action against ETX toxin challenge mice

In the second in vitro experiment, ZA can effectively inhibit the toxic effect of ETX toxin on MDCK cells, and is expected to be a good therapeutic drug for ETX toxin intoxication.

To verify this hypothesis, the study of the protective effect of ZA on ETX toxin-challenged animals was performed in vivo, and the specific experimental design was as follows:

1. lethality of ETX toxin to mice

6-week-old BALB/c male mice (purchased from Swiss Bei Fu (Beijing) Biotechnology Co., ltd., china) were selected for experiments, randomly divided into 5 groups, 5 mice were each group, and different concentrations of the above-mentioned ETX toxin GST-EXT (12800, 6400, 3200, 1600, 0 ng/kg) were intraperitoneally injected, respectively, and the states of the mice were observed for three consecutive days, and the survival rates thereof were recorded. The blank was injected with PBS buffer without ETX toxin (GST-EXT).

The results are shown in FIG. 5, where 12800ng/kg mice died completely within 3 hours, 6400ng/kg mice died completely within 8 hours, 3200ng/kg mice died 3 mice within 72 hours, and the remaining groups survived within 72 hours. Therefore, an ETX concentration of 6400ng/kg was chosen for subsequent experiments.

2. ZA inhibits lethality of ETX toxin to mice

BALB/c male mice 6 weeks old were selected for the experiment and randomly divided into 2 groups of 10 mice each:

50mg/kg ZA +6400ng/kg EXT group: intraperitoneally injecting 0.1ml ZA (50 mg/kg) every day in the first three days, and intraperitoneally injecting 0.1ml ETX toxin (6400 ng/kg) 30min after the injection of ZA in the third day;

6400ng/kg ETX group: within the first three days, 0.1ml PBS was intraperitoneally injected daily, and 30min after the injection of PBS on the third day, ETX toxin (6400 ng/kg) was intraperitoneally injected.

Subsequently, the status of the mice was observed for three consecutive days and their survival rate was recorded.

The experimental results are shown in fig. 6, compared with the ETX group, the addition of ZA to the ZA + EXT group can effectively reduce the toxic effect of ETX toxin on mice, significantly improve the survival rate of mice after ETX toxin challenge, and further show that ZA has a good therapeutic effect in animal bodies.

Therefore, ZA is expected to be an effective drug for preventing and treating ETX toxin, and a new idea is developed for the research of ETX toxin.

SEQUENCE LISTING

<110> military medical research institute of military science institute of people's liberation force of China

<120> application of Zaragozic acid A in treatment of clostridium perfringens Epsilon toxin poisoning

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 783

<212> DNA

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aaagcgtctt acgacaacgt ggacacgctg attgaaaaag gtcgttacaa cacgaaatac 60

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acgattaacc cgcagggtaa cgacttctac attaacaacc cgaaagtgga actggacggt 180

gaaccgagca tgaactatct ggaagacgtg tatgtgggta aagcgctgct gacgaacgac 240

acgcagcagg aacagaaact gaaaagccag agcttcacgt gcaaaaacac cgataccgtg 300

accgcgacca ccacccacac cgtgggcacc tcgatccagg caaccgctaa attcaccgtg 360

ccgtttaacg aaaccggcgt gagcctgacc accagctata gctttgcaaa caccaacacc 420

aacaccaaca gcaaagaaat tacccataac gtcccgagcc aggatatcct ggttccggct 480

aacaccaccg ttgaagttat cgcatatctg aaaaaagtta acgttaaagg taatgttaaa 540

ctggttggtc aggttagcgg tagcgaatgg ggtgaaatcc cgagctatct ggcttttccg 600

cgtgatggtt ataaatttag cctgagcgat accgttaata aaagcgatct gaatgaagat 660

ggtaccatta atattaatgg taaaggtaat tatagcgcag ttatgggtga tgaactgatc 720

gttaaagttc gtaatctgaa taccaataat gttcaggaat atgttatccc ggttgataaa 780

aag 783

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<212> PRT

<213> Artificial sequence

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Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro

1 5 10 15

Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu

20 25 30

Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu

35 40 45

Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys

50 55 60

Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn

65 70 75 80

Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu

85 90 95

Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser

100 105 110

Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu

115 120 125

Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn

130 135 140

Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp

145 150 155 160

Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu

165 170 175

Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr

180 185 190

Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala

195 200 205

Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg

210 215 220

Gly Ser Met Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys

225 230 235 240

Gly Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr

245 250 255

Tyr Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln

260 265 270

Gly Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu

275 280 285

Pro Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu

290 295 300

Thr Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr

305 310 315 320

Cys Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly

325 330 335

Thr Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr

340 345 350

Gly Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn

355 360 365

Thr Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu

370 375 380

Val Pro Ala Asn Thr Thr Val Glu Val Ile Ala Tyr Leu Lys Lys Val

385 390 395 400

Asn Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu

405 410 415

Trp Gly Glu Ile Pro Ser Tyr Leu Ala Phe Pro Arg Asp Gly Tyr Lys

420 425 430

Phe Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly

435 440 445

Thr Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp

450 455 460

Glu Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu

465 470 475 480

Tyr Val Ile Pro Val Asp Lys Lys

485

Claims (2)

1. The application of Zaragozic acid A in preparing a product with any one of the following functions:

   1) As an ETX toxin inhibitor;

   2) Preventing poisoning of the animal by the ETX toxin;

   3) Treating animal toxicity caused by ETX toxin;

   4) Inhibiting toxicity of ETX toxin;

   5) Neutralizing the ETX toxin;

   6) Reducing the toxic effect of the ETX toxin on cells;

   7) Improving the ability of the animal to resist the attack of ETX toxin;

   8) Reduce the animal lethality rate after the ETX toxin attack.
2. 2. The application of the substance taking Zaragozic acid A as an active ingredient in preparing a product with any one of the following functions:

   1) As an ETX toxin inhibitor;

   2) Preventing poisoning of the animal by the ETX toxin;

   3) Treating animal toxicity caused by ETX toxin;

   4) Inhibiting toxicity of ETX toxin;

   5) Neutralizing the ETX toxin;

   6) Reducing the toxic effect of the ETX toxin on cells;

   7) Improving the ability of the animal to resist the attack of ETX toxin;

   8) Reducing the animal mortality rate after the ETX toxin attack.

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