CN116407534A - Use of lipoic acid for treating diseases caused by clostridium perfringens Epsilon toxin - Google Patents

Abstract

The invention discloses the use of lipoic acid in the treatment of diseases caused by clostridium perfringens Epsilon toxin. In particular to the application of lipoic acid or derivatives thereof in preparing products for preventing, improving or treating diseases caused by clostridium perfringens infection. The invention verifies the inhibition effect of the lipoic acid on the Epsilon toxin through a cell level experiment, verifies the treatment effect of the lipoic acid on the Epsilon toxin poisoning through an animal experiment, and the result shows that the lipoic acid can effectively weaken the toxicity of the Epsilon toxin, delay the death time of animals poisoned by the Epsilon toxin, improve the survival rate of the animals poisoned by the Epsilon toxin, and has the purpose of protecting the animals from Epsilon toxin infection. The lipoic acid has good treatment effect on Epsilon toxin poisoning, and can be used as an ideal Epsilon toxin inhibitor for preventing or treating diseases caused by Epsilon.

Description

Lipoic acid in the treatment of diseases caused by Clostridium perfringens Epsilon toxin application

technical field

The invention belongs to the field of biotechnology, and in particular relates to the application of lipoic acid in the treatment of diseases caused by Clostridium perfringens Epsilon toxin.

Background technique

Clostridium perfringens (Clostridium perfringens), formerly known as Clostridium welchii, can produce various exotoxins such as α, β, γ, ε, θ, δ, η, ι, κ, λ, μ, ν, etc. According to the 6 main exotoxins α, β, ε, ι, CPE (enterotoxin) and NetB (pore forming toxin), they can be divided into seven types: A, B, C, D, E, F and G . Among them, Epsilon toxin (Epsilon toxin, ε toxin, ETX) is produced by Clostridium perfringens type B and type D and secreted into the host animal, which can cause fatal enterotoxemia in ruminants, and the disease is acute , rapid course of disease and high mortality rate, causing huge economic losses to animal husbandry every year. Epsilon toxin is a β-like pore-forming toxin that exists in the form of a heptamer. It can form a "β-barrel" structure composed of 14 β-sheets. This "β-barrel" structure can be inserted into the eukaryotic cells. The plasma membrane forms perforations. At the cellular level, Epsilon toxin can rapidly swell cell membranes, destroy various organelles, and eventually lead to necrosis of target cells. In mammals, Epsilon toxin can cause mammalian blood vessels to edema, thereby penetrating the blood-brain barrier and accumulating in the animal kidney and brain, causing the body to be overexcited with the release of glutamate. This series of reactions Occurrence can cause cerebral edema and renal failure in the body, eventually leading to the death of the animal. In addition, Epsilon toxin can not only cause multiple sclerosis in humans, but also specifically cause hemolysis of human red blood cells. Studies have found that the toxicity of Epsilon toxin is second only to botulinum toxin and tetanus toxin, and the US Centers for Disease Control and Prevention (CDC) has listed it as a Class B bioterrorist agent, which is a potential threat to human health and safety.

Animal kidney and brain are the main target organs of Epsilon toxin. When Epsilon toxin enters the animal body, it can accumulate in a large amount in the kidney and brain, causing congestion and edema, destroying the blood-brain barrier, and causing animal death. However, there are not many cells sensitive to Epsilon toxin, mainly mouse renal cortical collecting duct (mpkCCDcl4) cells, human renal leiomyoma (G-402) cells, Fisher rat thyroid (FRT) cells and human renal granulosa cell carcinoma (ACHN) cells, the most sensitive of which are canine kidney epithelial (MDCK) cells. When Epsilon toxin attacks cells, it first binds to specific receptors on the cell surface, forms a heptamer complex on lipid rafts, and forms a large pore on the surface of the cell membrane, resulting in a change in the permeability of the cell membrane and a large amount of toxic substances entering the cell , eventually causing cell death.

At present, the treatment of Epsilon toxin poisoning mainly uses antibodies to neutralize Epsilon toxin, and most of them are in the research stage, and there is still a lack of effective therapeutic drugs in clinical practice. Therefore, it is necessary to research and develop safe and effective new Epsilon toxin poisoning treatment drugs and methods. , has wide clinical application value and important significance to the prevention and treatment of diseases caused by Clostridium perfringens Epsilon toxin.

Contents of the invention

The technical problem to be solved by this invention is that in the prior art, there is a lack of effective therapeutic drugs for Epsilon toxin poisoning, and a kind of lipoic acid or its derivatives is provided in the preparation of medicines for the treatment of diseases caused by Clostridium perfringens Epsilon toxin. application. The technical problems to be solved are not limited to the described technical subjects, and those skilled in the art can clearly understand other technical subjects not mentioned herein through the following description.

In order to solve the above technical problems, the present invention firstly provides the application of lipoic acid or its derivatives in the preparation of products for preventing, improving or treating diseases caused by Clostridium perfringens infection.

The molecular structural formula of the lipoic acid (Thioctic acid, CAS No. 1077-28-7) is shown in FIG. 2 .

In the above application, the Clostridium perfringens may be type B and/or type D Clostridium perfringens.

In the above application, the disease caused by Clostridium perfringens infection may be a disease caused by Clostridium perfringens Epsilon toxin (also known as epsilon toxin, Epsilon toxin, ETX).

The amino acid sequence of the Epsilon toxin may be 228-488 of SEQ ID No.2, and the nucleotide sequence of the gene encoding the Epsilon toxin may be SEQ ID No.1.

In the above application, the diseases caused by Clostridium perfringens Epsilon toxin include but not limited to Clostridium perfringens Epsilon toxin poisoning, enterotoxemia, necrotic enteritis, nephropathy, intestinal disease, brain disease ( such as encephalitis or meningitis) or multiple sclerosis.

Symptoms of the disease caused by the Clostridium perfringens Epsilon toxin may include edema of the main target organs of the kidney and brain and damage of vascular endothelial cells.

In the above application, the product may have at least any of the following functions:

A1) preventing, improving or treating diseases caused by Clostridium perfringens infection;

A2) preventing, improving or treating diseases caused by Type B and/or Type D Clostridium perfringens infection;

A3) preventing, improving or treating diseases caused by Clostridium perfringens Epsilon toxin;

A4) Neutralize Clostridium perfringens Epsilon toxin;

A5) reducing or inhibiting the toxic effect of Clostridium perfringens Epsilon toxin;

A6) improve the ability of animals to resist the attack of Clostridium perfringens Epsilon toxin;

A7) improve the survival rate of animals poisoned by Clostridium perfringens Epsilon toxin;

A8) as Clostridium perfringens Epsilon toxin inhibitor;

A9) As an anti-Clostridium perfringens agent.

The present invention also provides any of the following applications of substances with lipoic acid and/or derivatives thereof as active ingredients:

B1) Application in the preparation of products for preventing, improving or treating diseases caused by Clostridium perfringens infection;

B2) Application in the preparation of products for preventing, improving or treating diseases caused by Type B and/or Type D Clostridium perfringens infection;

B3) Application in the preparation of products for preventing, improving or treating diseases caused by Clostridium perfringens Epsilon toxin;

B4) Use in the preparation of a product for neutralizing Clostridium perfringens Epsilon toxin;

B5) Application in the preparation of products for reducing or inhibiting the toxic effect of Clostridium perfringens Epsilon toxin;

B6) Application in the preparation of products for improving the ability of animals to resist the attack of Clostridium perfringens Epsilon toxin;

B7) Application in the preparation of products for improving the survival rate of animals poisoned by Clostridium perfringens Epsilon toxin;

B8) application in the preparation of Clostridium perfringens Epsilon toxin inhibitor;

B9) Application in the preparation of an anti-Clostridium perfringens preparation.

Animals described herein include humans and non-human animals.

A product described herein may be a drug or an agent.

In the above applications, the substance can be a pharmaceutical composition, feed or feed additive.

In the above applications, the feed or feed additive can be used for ruminants, poultry, pigs, horses, rabbits or mice.

Further, the ruminants may include cattle (such as cattle, yaks, cows or calves, etc.) or sheep (such as sheep, goats, or lambs, etc.), and the poultry may include chickens, ducks or geese.

Further, the pharmaceutical composition also includes one or more pharmaceutically acceptable carriers.

The pharmaceutically acceptable carrier may be a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorption carrier, surfactant or lubricant, but is not limited thereto.

The present invention mainly has two aspects: one is to verify the inhibitory effect of lipoic acid on Epsilon toxin at the cell level. Based on previous work accumulation, the inventors verified the inhibitory effect of lipoic acid on the toxicity of Epsilon toxin in MDCK cells. The results showed that lipoic acid could effectively inhibit the toxic effect of Epsilon toxin on MDCK cells in a concentration-dependent manner. Therefore, the present invention shows that lipoic acid has an important application prospect in the treatment of Epsilon toxin poisoning.

Another aspect of the present invention is to verify the treatment effect of lipoic acid on animal poisoning caused by Epsilon toxin through animal experiments. Applying lipoic acid can effectively weaken the toxicity of Epsilon toxin, and improve the survival rate of animals poisoned by Epsilon toxin, and has the purpose of protecting animals against Epsilon toxin infection. Therefore, lipoic acid can be used in the preparation of related drugs or reagents for preventing or treating Epsilon toxin poisoning in animals.

The lipoic acid involved in the present invention has a clear molecular structure and is easy to obtain. The preparation method of the Epsilon toxin involved in the present invention is relatively mature, and is mainly obtained through recombinant expression. Epsilon toxin is the most sensitive to MDCK cells, so it is used in the research on the treatment of lipoic acid poisoning in the present invention. This validation method falls within the category of classical methods in the field of toxin research.

Lipoic acid showed inhibitory effect on the toxicity of Epsilon toxin in MDCK cytotoxicity experiment and mouse challenge experiment, which showed that the survival rate of MDCK cells was improved, and the mice challenged with a completely lethal dose of Epsilon toxin were treated with lipoic acid. , the survival rate was significantly improved. It is disclosed in the present invention that lipoic acid has a good therapeutic effect on Epsilon toxin poisoning, has a good application prospect in animal husbandry, and is of great significance to the biological safety of human society.

The beneficial effects of the present invention are:

1. The lipoic acid involved in the present invention has a good neutralizing effect on the toxicity of Epsilon toxin. At the cellular level, lipoic acid has a better inhibitory effect on the toxicity of Epsilon toxin. In animal experiments, lipoic acid showed a certain protective effect on animals challenged with Epsilon toxin. Therefore, lipoic acid is expected to become an important reagent and drug for the prevention and treatment of infections caused by Epsilon toxin.

2. The lipoic acid designed by the present invention has a simple molecular structure and is easy to obtain on the market. It has a good application prospect in animal husbandry and is of great significance to the safety of human society.

The inventor has been engaged in the research work related to Epsilon toxin for a long time, and found that lipoic acid (Thioctic acid, CAS No. 1077-28-7) can weaken the toxic effect of Epsilon toxin on cells, and can protect mice against lethal dose of Epsilon toxin to a certain extent. Attack, improve survival rate.

Description of drawings

Figure 1 is the result of SDS-PAGE after purification of Epsilon toxin.

Figure 2 is a molecular structure diagram of lipoic acid.

Figure 3 is a graph showing the cytotoxicity results of different concentrations of Epsilon toxin on MDCK cells.

Fig. 4 is a diagram showing the results of lipoic acid inhibiting the cytotoxicity of Epsilon toxin to MDCK.

Figure 5 is a graph showing the survival curves of mice challenged with different concentrations of Epsilon toxin.

Fig. 6 is a survival curve of mice challenged with Epsilon toxin by lipoic acid.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guideline for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The molecular structural formula of lipoic acid (Thioctic acid, CAS No. 1077-28-7) in the following examples is shown in FIG. 2 .

The pGEX-4T-1 vector in the following examples is a product of Cytiva Company, the product catalog number is: 28954549.

The MDCK cells in the following examples are products of Wuhan Puruosai Life Technology Co., Ltd., and the product catalog number is: CL-0154.

Embodiment 1, the application of lipoic acid in inhibiting Epsilon toxin

1. Expression and purification of Epsilon toxin

1. Construction of Epsilon toxin recombinant strain BL21(DE3)

The present invention uses the Epsilon toxin recombinant strain BL21 (DE3) preserved in our laboratory to purify and obtain Epsilon toxin with higher purity for subsequent research. Epsilon toxin recombinant strain BL21(DE3) is a recombinant bacterium obtained by introducing recombinant vector pGEX-4T-1-Epsilon into Escherichia coli BL21(DE3). The construction method of Epsilon toxin recombinant strain BL21 (DE3) is as follows:

1.1. Construction of recombinant vector pGEX-4T-1-Epsilon

The recombinant vector pGEX-4T-1-Epsilon replaces the fragment (small fragment) between the BamHI and XhoI recognition sites of the pGEX-4T-1 vector with the DNA fragment of SEQ ID No.1 in the sequence listing (ie the Epsilon toxin gene) , keeping other nucleotide sequences of the pGEX-4T-1 vector unchanged to obtain a recombinant expression vector. The Epsilon toxin gene and the GST tag on the carrier are fused and expressed to obtain the Epsilon toxin that has fused the GST tag. The amino acid sequence of the Epsilon toxin that has fused the GST tag is SEQ ID No.2, wherein, the 228th- of SEQ ID No.2 Position 488 is Epsilon toxin (encoded by the Epsilon toxin gene shown in SEQ ID No.1), and positions 1-226 are GST tags.

1.2. Construction of Epsilon toxin recombinant strain BL21(DE3)

Transform the plasmid (recombinant vector pGEX-4T-1-Epsilon) constructed in the above step 1.1 into Escherichia coli BL21 (DE3) competent cells, screen and identify the transformed colonies, and obtain the recombinant bacteria that correctly insert the plasmid, which is the Epsilon toxin recombinant strain BL21(DE3), store at -80°C for future use.

2. Expression and purification of Epsilon toxin

2.1. Cultivation and induction of Epsilon toxin recombinant strain BL21(DE3)

Take out the preserved Epsilon toxin recombinant strain BL21(DE3) from the -80°C refrigerator, and transfer it into 5ml LB liquid medium containing 100μg/ml ampicillin at a ratio of 1:100 (500ml medium needs to be added with 5g peptone, 2.5 g yeast extract and 5g NaCl, the balance being water), cultured at 180r/min at 37°C for 6h, then transferred to 500ml LB liquid medium containing 100μg/ml ampicillin at a ratio of 1:100, and incubated at 180r/min at 37°C Shake culture at 1/min until the OD ₆₀₀ value is between 0.6-0.8, add IPTG to a final concentration of 1 mM to induce the expression of exogenous genes, and culture overnight at 16°C at 180 r/min to obtain a culture solution.

2.2. Preparation of protein mixture

Centrifuge the culture medium obtained in the above step 2.1 at 4°C for 15 minutes at 8000r/min to collect the bacteria, discard the supernatant, resuspend the bacteria in PBS, centrifuge at 8000r/min for 15min, discard the supernatant, and use 80ml of liquid A (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 water should be added to 1LA solution), resuspend the bacteria, ultrasonicate (power 75 %, break 3s, pause 2s). At 4°C, centrifuge the broken bacterial solution at 12,000 r/min for 15 minutes, filter the supernatant with a 045 μm filter, and set aside for later use to obtain a protein mixture.

2.3. Purification of Epsilon toxin by purifier

(1) Rinse the pumps A and B of the purification apparatus with sterile water at a flow rate of 3ml/min;

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

(3) Install the GSTrap ^™ HP chromatography column (Cytiva Company, catalog number: 17528201) on the purifier, and wash the A pump and the GSTrap ^™ HP chromatography column with liquid A at a flow rate of 3ml/min;

(4) Load the protein mixture prepared in step 2.2 through the GSTrap ^TM HP chromatography column treated in (3) at a flow rate of 1ml/min;

(5) Rinse the GSTrap ^TM HP chromatography column with solution A at a flow rate of 1ml/min;

(6) Use solution B to elute the protein bound to the GSTrap ^TM HP chromatography column in a gradient manner at a flow rate of 1 ml/min, and collect the effluent from the elution peak for SDS-PAGE analysis.

The results are shown in Figure 1, the breakthrough liquid is the product collected in the above step (4); liquid A is the product collected in the above step (5), and liquid B is the product collected in the above step (6); it can be seen that after purification A highly pure Epsilon toxin fused with a GST tag (named GST-Epsilon) was obtained, with a size of 56 kDa, an amino acid sequence of SEQ ID No.2, and a concentration of 4 mg/ml, which can be used for subsequent experiments.

The Epsilon toxins used in the following experiments are all GST-Epsilon.

2. Verification of the ability of lipoic acid to inhibit the cytotoxicity of Epsilon toxin on MDCK cells

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

The invention uses the MTS assay method to verify the inhibition of lipoic acid on the death of MDCK cells caused by Epsilon toxin, as an evaluation index for verifying the inhibitory effect of lipoic acid on the toxicity of Epsilon toxin. MDCK cells were diluted to 10 ⁵ cells/ml with DMEM medium containing 10% (volume percentage) FBS before use, spread in a 96-well plate, each well contained 10 ⁴ cells, and cultured in a cell culture incubator for 24 hours for use .

2. Determination of the median lethal dose of Epsilon toxin to MDCK cells

Aqueous One Solution CellProliferation Assay Kit，Promega公司产品，产品目录号：G3581)测定细胞存活率，以确定Epsilon毒素对MDCK细胞半数致死剂量，步骤如下：Using the MTS assay method, the MTS test kit (

Aqueous One Solution CellProliferation Assay Kit, Promega company product, product catalog number: G3581) measure cell viability, to determine the half lethal dose of Epsilon toxin to MDCK cells, the steps are as follows:

Experimental group: Dilute the Epsilon toxin (GST-Epsilon) prepared in the above one 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, a total of 18 gradients were added to the 96-well plate according to the volume of 100μl/well.

Blank control group: only 100 μl/well of DMEM medium containing 10% (volume percentage) FBS was added as a control group.

After the above groups were incubated in the cell culture incubator for 1 h, discard the culture medium, wash with PBS for 3 times, then add 100 μl/well of DMEM medium containing 10% (volume percentage) FBS and 20 μl/well of MTS, in the cell culture incubator After incubation in medium for 3 h, the absorbance value at 492 nm was detected, and the survival rate of MDCK cells was calculated according to the following formula.

Cell survival rate (%) = absorbance value at 492nm of the experimental group/absorbance value at 492nm of the control group × 100

The median lethal dose of Epsilon toxin to MDCK cells was further determined through the Epsilon concentration-cell survival rate curve, and the results are shown in Figure 3. After calculation, the median lethal dose of Epsilon toxin to MDCK cells was about 100 ng/ml.

3. Incubation of different concentrations of lipoic acid and Epsilon toxin

Use 150 μ l DMSO to dissolve 150 mg lipoic acid (Selleck, the United States, product number S3996), and then use DMEM medium containing 10% (volume percentage) FBS to dilute it to 66, 33, 16.5, 8.3, 4.1, 2, 0 mg /ml, a total of 7 gradients, respectively diluted with an equal volume of DMEM medium containing 10% (volume percentage) FBS to a final concentration of 100ng/ml Epsilon toxin mix, placed in a 37 ° C water bath for 30min incubation, to obtain Epsilon A mixture of toxins and lipoic acid.

4. Epsilon toxin attacks cells

Experimental group: add the mixture of Epsilon toxin and lipoic acid prepared in the above step 3 into the 96-well cell plate of MDCK cells prepared in the above step 1 according to the volume of 100 μl/well;

Control group: the Epsilon toxin prepared above was diluted with DMEM medium containing 10% (volume percentage) FBS to a final concentration of 100 ng/ml, and added to a 96-well plate with only MDCK cells in a volume of 100 μl/well.

After incubating in the cell culture incubator for 1 h, discard the medium, wash with PBS 3 times, then add 100 μl/well of DMEM medium containing 10% (volume percentage) FBS and 20 μl/well of MTS, and incubate in the cell incubator for 3 h , detect the absorbance value at 492nm, and calculate the cell survival rate according to the formula in the above step 2.

Lipoic acid inhibits the cytotoxicity of Epsilon toxin on MDCK cells, as shown in Figure 4. The results show that lipoic acid has a significant inhibitory effect on MDCK cytotoxicity caused by Epsilon toxin, and effectively improves the survival rate of MDCK cells caused by Epsilon toxin.

3. Protective effect of lipoic acid on mice challenged with Epsilon toxin

In the in vitro experiment of the previous step 2, lipoic acid can effectively inhibit the toxic effect of Epsilon toxin on MDCK cells, and it is speculated that it is expected to become a good drug for the treatment of Epsilon toxin poisoning.

In order to verify this conjecture, it is planned to carry out a study on the protective effect of lipoic acid on animals challenged with Epsilon toxin in animals, and the specific experimental design is as follows:

1. Lethality of Epsilon toxin in mice

In the experiment, 6-week-old BALB/c male mice (purchased from Speifu (Beijing) Biotechnology Co., Ltd., China) were randomly divided into 5 groups, 5 mice in each group, and different concentrations (12800, 6400 , 3200, 1600, 0ng/kg) of the 0.1ml/Epsilon toxin prepared in the above step one, observe the state of the mice for three consecutive days, and record their survival rate. When the Epsilon toxin is 0ng/kg, it means that only an equal volume of PBS is injected, which is used as a blank control.

The results are shown in Figure 5. All the mice in the 12800ng/kg group died within 3 hours, all the mice in the 6400ng/kg group died within 8 hours, 3 mice in the 3200ng/kg group died within 72 hours, and the other groups died within 72 hours. Both survived. Therefore, 6400ng/kg of Epsilon toxin was selected for subsequent experiments.

2. Lipoic acid inhibits the lethality of Epsilon toxin in mice

In the experiment, 6-week-old BALB/c male mice were randomly divided into 3 groups, with 10 mice in each group, which were:

400mg/kg lipoic acid+6400ng/kg Epsilon toxin group: intraperitoneal injection of lipoic acid (400mg/kg), 0.1ml/rat, 30min later, intraperitoneal injection of Epsilon toxin (6400ng/kg), 0.1ml/rat;

6400ng/kg Epsilon toxin+400mg/kg lipoic acid group: intraperitoneal injection of Epsilon toxin (6400ng/kg), 0.1ml/rat, 30min later, intraperitoneal injection of lipoic acid (400mg/kg), 0.1ml/rat;

6400ng/kg Epsilon toxin group: intraperitoneal injection of ETX toxin (6400ng/kg), 0.1ml/rat, 30min later, intraperitoneal injection of PBS, 0.1ml/rat.

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

The experimental results are shown in Figure 6. Compared with the Epsilon toxin group (8h survival rate dropped to 0), the addition of lipoic acid to the lipoic acid+Epsilon toxin group can effectively delay the death time of mice after Epsilon toxin challenge, and can improve Survival rate of mice challenged with Epsilon toxin. At the same time, it further shows that lipoic acid also has a certain therapeutic effect in animals.

In conclusion, whether it is in vitro cell experiments or in vivo mouse experiments, the experimental results show that lipoic acid can effectively reduce or inhibit the toxicity of Epsilon toxin, improve the ability of animals to resist Epsilon toxin attack, and can be used as an ideal Epsilon toxin inhibitor. Agents are used to prevent or treat diseases caused by Epsilon toxins. Therefore, lipoic acid is expected to become an effective drug for the prevention and treatment of diseases related to Epsilon toxin poisoning, has good clinical application prospects, and opens up new ideas for the research of Epsilon toxin.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application. Applications of some of the essential features are possible within the scope of the appended claims below.

Claims (8)

1. Application of lipoic acid or derivatives thereof in the preparation of products for preventing, improving or treating diseases caused by Clostridium perfringens infection. 2. The application according to claim 1, characterized in that the Clostridium perfringens is type B and/or type D Clostridium perfringens. 3. The application according to claim 1 or 2, characterized in that the disease caused by Clostridium perfringens infection is a disease caused by Clostridium perfringens Epsilon toxin. 4. The application according to claim 3, wherein the diseases caused by the Clostridium perfringens Epsilon toxin include but not limited to Clostridium perfringens Epsilon toxin poisoning, enterotoxemia, necrotic enteritis , kidney disease, bowel disease, brain disease, or multiple sclerosis. 5. The application according to any one of claims 1-4, wherein the product has at least one of the following functions: A1) preventing, improving or treating diseases caused by Clostridium perfringens infection; A2) preventing, improving or treating diseases caused by Type B and/or Type D Clostridium perfringens infection; A3) preventing, improving or treating diseases caused by Clostridium perfringens Epsilon toxin; A4) Neutralize Clostridium perfringens Epsilon toxin; A5) reducing or inhibiting the toxic effect of Clostridium perfringens Epsilon toxin; A6) improve the ability of animals to resist the attack of Clostridium perfringens Epsilon toxin; A7) improve the survival rate of animals poisoned by Clostridium perfringens Epsilon toxin; A8) as Clostridium perfringens Epsilon toxin inhibitor; A9) As an anti-Clostridium perfringens agent. 6. Any of the following applications of substances with lipoic acid and/or its derivatives as active ingredients: B1) Application in the preparation of products for preventing, improving or treating diseases caused by Clostridium perfringens infection; B2) Application in the preparation of products for preventing, improving or treating diseases caused by Type B and/or Type D Clostridium perfringens infection; B3) Application in the preparation of products for preventing, improving or treating diseases caused by Clostridium perfringens Epsilon toxin; B4) Use in the preparation of a product for neutralizing Clostridium perfringens Epsilon toxin; B5) Application in the preparation of products for reducing or inhibiting the toxic effect of Clostridium perfringens Epsilon toxin; B6) Application in the preparation of products for improving the ability of animals to resist the attack of Clostridium perfringens Epsilon toxin; B7) Application in the preparation of products for improving the survival rate of animals poisoned by Clostridium perfringens Epsilon toxin; B8) application in the preparation of Clostridium perfringens Epsilon toxin inhibitor; B9) Application in the preparation of an anti-Clostridium perfringens preparation. 7. The application according to claim 6, characterized in that the substance is a pharmaceutical composition, feed or feed additive. 8. The use according to claim 7, characterized in that the feed or feed additive is used for ruminants, poultry, pigs, horses, rabbits or mice.

Images