CN112294796A - Application of anwurtzitin in resisting aquatic animal aeromonas hydrophila infection - Google Patents
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Abstract
The invention discloses application of anwurtzitin in resisting aquatic animal aeromonas hydrophila infection, the anwurtzitin has no influence on the growth of the aquatic aeromonas hydrophila, but can reduce the pathogenicity of the aquatic aeromonas hydrophila by inhibiting the expression of the aquatic aerolysin, and further proves the in vivo treatment effect of the aquatic aeromonas hydrophila infection by establishing an animal infection model. The anwuweisu is one of chemical components of Chinese medicine schisandra fruit, has several biological activities and has no toxicity to animal and human body, so that it may be prepared into medicine preparation or feed additive for preventing and treating aeromonas hydrophila disease of aquatic animal.
Description
Technical Field
The invention belongs to the technical field of aquaculture, and particularly relates to application of anwurtzitane in resisting aquatic animal aeromonas hydrophila infection.
Background
The schisandra chinensis is also named as fructus schisandrae, zanthoxylum bungeanum and the like, is a dry fruit of schisandra chinensis of the magnoliaceae, schisandra genus, is listed as a top-grade traditional Chinese medicinal material in Shennong herbal channels, is a famous traditional Chinese medicine, has warm property, sweet and sour taste, and has the effects of astringing, arresting discharge, benefiting qi, promoting the production of body fluid, calming heart and tonifying kidney. It is mainly used for treating lung deficiency, cough and asthma, spontaneous perspiration and night sweat. Modern medicine research shows that the schisandra contains various chemical components such as schizandrin, schizandrol, schisantherin and the like, and the extract of the schisandra has pharmacological effects of protecting liver, inhibiting central nervous system, strengthening heart, reducing blood pressure, enhancing immunity, resisting bacteria and the like; can be used for treating icterohepatitis, acute intestinal infection, neurasthenia, and bacterial cystitis.
Aeromonas hydrophila is a gram-negative bacterium, widely distributed in natural environment, and is a common bacterium in aquaculture water. The bacterium is a opportunistic pathogen that can infect terrestrial animals, aquatic animals and humans. Aeromonas hydrophila in freshwater aquaculture often causes fulminant hemorrhagic disease after infecting aquatic animals, can infect most aquatic animals, is a fulminating infectious disease of pond culture, and often causes higher mortality and serious economic loss. Chemical antibacterial drugs are the main means used by the aquaculture industry for treating bacterial diseases, but due to the unreasonable use of antibacterial drugs, the problem of drug resistance is extremely serious. At present, a large number of drug-resistant aeromonas hydrophila strains insensitive to antibacterial drugs are reported at home and abroad, which brings great difficulty to the treatment of the aquatic animal aeromonas hydrophila disease. In addition, the dosage of the antibacterial agent is inevitably increased in order to control diseases caused by drug-resistant strains, so that the antibacterial agent is accumulated and remained in aquatic animal food, and potential influence is brought to the quality and safety of the food. In conclusion, the screening of the medicine with a brand-new anti-infection mechanism has important significance for guaranteeing the healthy development of the aquaculture industry.
Aerolysin is one of the important virulence factors secreted by Aeromonas hydrophila and is a marker for identifying pathogenic Aeromonas hydrophila. Research finds that the pathogenicity of the aerolysin gene knocked out by the aeromonas hydrophila is obviously reduced, and the aerolysin plays a key role in the pathogenic process of the aeromonas hydrophila and can be used as a target for research and development of new drugs.
Disclosure of Invention
The invention aims to provide application of the anwurtzitin resisting aeromonas hydrophila infection of aquatic animals, wherein the anwurtzitin reduces the expression of aerolysin by inhibiting the transcription of an aerolysin coding gene, thereby realizing the effect of reducing the pathogenicity of aquatic animals.
In order to achieve the purpose, the invention adopts the following technical scheme:
the application of the anwurtzitin resisting aeromonas hydrophila infection of the aquatic animals comprises the following steps: experiments prove that the immunophilin can obviously inhibit the hemolytic activity of the supernatant of the aeromonas hydrophila co-culture and obviously inhibit the transcription of the aerolysin coding gene aerA when the concentration of the immunophilin reaches more than 2 mug/mL; the administration dosage of the amphetamine to the channel catfish infected with the aeromonas hydrophila is 20mg/kg, the continuous use is carried out for 3 days, and the survival rate after the treatment can reach more than 60 percent.
The anwuweisu is one of the chemical components of the Chinese traditional medicine schisandra chinensis, and researches show that the anwuweisu has no toxic or side effect on animals and human beings, is environment-friendly, and can be used as a pharmaceutical preparation or a feed additive for preventing or treating aeromonas hydrophila infection of aquatic animals.
Compared with the prior art, the invention has the following advantages and effects: the anwuqin does not directly sterilize, but reduces the pathogenicity of the anwuqin, and drug resistance is not easy to generate; the anwurtzitin directly acts on the expression of the aerolysin and has a treatment effect on drug-resistant aeromonas hydrophila; the anwurtzitane is a natural compound, is environment-friendly, has no toxicity to animals, and reduces the risk of drug residue.
Drawings
FIG. 1 is a graph showing the effect of anwurtzitol on the growth of Aeromonas hydrophila
FIG. 2 is a graph showing the effect of anwurtzitol on the hemolytic activity of Aeromonas hydrophila culture supernatant
FIG. 3 is a graph showing the effect of anwurtzitin on transcription of Aeromonas hydrophila aerolysin coding gene aerA
Detailed Description
Example 1: effect of anwuxidin on Aeromonas hydrophila growth
The aeromonas hydrophila XS-91-4-1 strain related in the embodiment is stored in a laboratory of aquatic animal pharmacology and drug residue control technology of Yangtze river aquatic product research institute. XS-91-4-1 strain was cultured in BHI broth to logarithmic growth phase (OD)600nm0.3, 28 ℃), subpackaging the cultured bacteria into 6 conical flasks with 50mL, adding different concentrations (0, 1, 2, 4, 8 mu g/mL) of the anwurtzitaxel (Szechwan Biotech limited) into 20mL conical flasks, culturing for 5h at 28 ℃, taking the bacteria liquid every 30min, and measuring OD (optical density) of the bacteria liquid600nmThe absorption value of (2). FIG. 1 shows the growth curves of different concentrations of ampelopsin co-cultured with Aeromonas hydrophila XS-91-4-1, from which it can be seen that ampelopsin had no effect on the growth of Aeromonas hydrophila at the concentrations tested.
Example 2: effect of anwuliptin on hemolytic Activity of Aeromonas hydrophila supernatant
Aeromonas hydrophila XS-91-4-1 was cultured overnight in LB liquid medium. The following day is as follows: 100 percent of the culture medium is transferred into 200mL of fresh LB liquid culture medium, and the culture is continued until OD600nmAbout 0.3 hours, 5 bottles were dispensed, and anwurtzitone was added to a concentration of 8. mu.g/mL, 4. mu.g/mL, 2. mu.g/mL, 1. mu.g/mL, and 0. mu.g/mL. Continuing to culture until OD600nmThe bacterial solution was collected at 1.5, centrifuged at 12000rpm for 1min and the supernatant was assayed for hemolytic activity. The method comprises the following steps: adding 875 μ L hemolysis buffer (20mM Tris 7.2, 0.9% NaCl), 100 μ L bacteria liquid supernatant, 25 μ L defibrinated sheep red blood cell into 1.5mL centrifuge tube, mixing, placing into 37 deg.C incubator, standing, incubating for 15min, centrifuging at 12000rpm, and determining OD of supernatant543nmThe absorption value of (2). As shown in fig. 2, the hemolysis of the supernatant gradually decreased with increasing concentration of anwurtzitane, and was dose-dependent. The test of hemolysis shows that the ampanolipin can reduce the hemolytic activity of the supernatant of aeromonas hydrophila, and the suggestion that the medicine can act on aerolysinExpression of (2).
Example 3: effect of anwurtzitin on Aerosol coding Gene aerA transcription
Culturing Aeromonas hydrophila XS-91-4-1 in LB liquid culture medium to logarithmic growth prophase (OD)600nm0.3, 28 ℃ C.), the culture medium is placed in 5 conical flasks of 50mL, 20mL of the culture medium is added with the ampanolide with the concentration of 8, 4, 2, 1, 0. mu.g/mL, and the culture is continued at 28 ℃ until OD is reached600nmThe cells were collected by high speed centrifugation (8000g,1min) until 1.5 hours. The thalli adopts a total RNA extraction kit of rhizobacteria to extract thalli RNA, adopts a Takara RNA PCR kit (AMV) kit to synthesize cDNA, adopts a SYBR Premix Ex Taq kit to carry out a fluorescent quantitative PCR test, analyzes the expression level of an aerosol encoding gene aerA by a delta Ct method, and takes 16s rRNA as an internal reference. 3 independent replicates were performed for each drug concentration group. The primers used in the assay were synthesized by Shanghai Biotech, and the sequences were as follows: aerA gene upstream primer (aerA-F): 5'-TCTACCACCACCTCCCTGTC-3', downstream primer (aerA-R): 5'-GACGAAGGTGTGGTTCCAGT-3', the molecular weight of the amplified product is 218 bp. 16s rRNA upstream primer (16s rRNA-F): 5' -TAATACCGCATACGCCCTAC-3, downstream primer (16s rRNA-R): 5'-ACCGTGTCTCAGTTCCAGTG-3', the molecular weight of the amplified product is 164 bp. As shown in figure 3, the transcription of the aerosol encoding gene aerA can be remarkably inhibited after the concentration of the anwurtziten reaches more than 2 mug/mL, and the inhibition effect is dose-dependent. This result suggests that anwurtzitin reduces the expression level of aerolysin in the culture supernatant by inhibiting transcription of aerA.
Example 4: therapeutic effect of anwuliptin on model infected by aeromonas hydrophila of channel catfish
The 60 healthy channel catfishes (from the fishing medicine clinical test center of Yangtze river aquatic research institute) are respectively placed in 3 200L glass jars, 20 channels of each group are kept at the water temperature of 28 ℃, the dissolved oxygen is 5.5-5.7mg/L, and the channel catfishes are temporarily raised for 7 days before toxicity attack. The aeromonas hydrophila XS-91-4-1 strain is cultured in an LB culture medium to reach the middle logarithmic growth phase (OD)600nm1.0), the bacterial solution was centrifuged, the cells were washed 2 times with sterile PBS and resuspended to 1.5 × 10 using a turbidimetric tube8CFU/mL, each of positive control group and treatment group200 mu L of diluted bacteria solution is injected into the abdominal cavity of the fish, and 200 mu L of sterile PBS is injected into the abdominal cavity of the negative control group. The treatment groups were orally infused with 20mg/kg of anwuxitin 6h after infection, once every 12h, continuously administered for three days, and the death status of each group of fish was observed and recorded every day. As shown in Table 1, the death rate of the channel catfish infected with Aeromonas hydrophila within 5 days without drug treatment is 90%, and the death rate after 20mg/kg of the treatment of the pentadecanol is 35%, which is lower than that without drug treatment. The results show that the anwurtzitaxenic can be used for treating the aeromonas hydrophila infection of the channel catfish, the dosage of the anwurtzitaxenic is 20mg/kg, the administration is carried out once every 12h, and the continuous use is carried out for 3 days.
TABLE 1 therapeutic Effect of anwurtzitin on model of Aeromonas hydrophila infection of Ictalurus punctatus
And (3) knotting:
it was found by example 1 that anwurtzitol had no effect on the growth of the Aeromonas hydrophila XS-91-4-1 strain at the concentrations tested, suggesting that anwurtzitol had no bacteriostatic effect on Aeromonas hydrophila at and below this concentration. It was found by example 2 that anwurtzitane dose-dependently reduced the hemolytic activity of the culture supernatant of Aeromonas hydrophila, suggesting that anwurtzitane inhibited the expression or activity of aerolysin. Further, it was found from example 3 that anwurtzitin inhibited the transcription of the gene aerA encoding aerolysin, thereby reducing the expression level of aerolysin in the supernatant. Finally, the study of example 4 shows that 20mg/kg of the anwurtzitane can reduce the death rate of the model of the channel catfish aeromonas hydrophila, and the anwurtzitane can be used as a medicine for treating the channel catfish aeromonas hydrophila infection.
Aeromonas hydrophila is a conditioned pathogen widely existing in aquaculture environment, and can cause diseases of most freshwater aquaculture animals, such as silver carps, grass carps, Chinese soft-shelled turtles, procambarus clarkia and the like. Aerolysin is the material basis for a variety of infections caused by Aeromonas hydrophila, and is prevalent among pathogenic strains of Aeromonas hydrophila. It can be concluded that the anwurtzitol has therapeutic effect on aeromonas hydrophila infection of aquaculture animals.
Claims (5)
1. Application of anwuxitin in preparing medicine for inhibiting expression of aerolysin of Aeromonas hydrophila is provided.
2. Application of anwuqin in preparing medicine for preventing or treating aeromonas hydrophila infection of aquatic animals is provided.
3. The use of claim 2 wherein the amount of anwurtzitaxel administered is greater than 20 mg/kg.
4. A medicament for combating aeromonas hydrophila infection in aquaculture animals, which comprises an anwurtzitane.
5. An aquatic feed additive for resisting aeromonas hydrophila infection is characterized in that the additive is anwurtzitan.
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| CN202011235819.8A CN112294796B (en) | 2020-11-06 | 2020-11-06 | Application of ampentaipine in resisting aeromonas hydrophila infection of aquatic animals |
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| CN202011235819.8A CN112294796B (en) | 2020-11-06 | 2020-11-06 | Application of ampentaipine in resisting aeromonas hydrophila infection of aquatic animals |
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| CN112294796A true CN112294796A (en) | 2021-02-02 |
| CN112294796B CN112294796B (en) | 2023-04-21 |
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| CN202011235819.8A Active CN112294796B (en) | 2020-11-06 | 2020-11-06 | Application of ampentaipine in resisting aeromonas hydrophila infection of aquatic animals |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006104369A1 (en) * | 2005-03-31 | 2006-10-05 | Amicogen, Inc | Novel use of lignan compounds |
| CN101410100A (en) * | 2004-01-08 | 2009-04-15 | 黄在宽 | Method and composition for treating acne using lignan compounds |
| CN105708926A (en) * | 2016-01-28 | 2016-06-29 | 西南大学 | Compound Chinese herbal medicine for inhibiting and killing aeromonas hydrophila as well as preparation method and application of compound Chinese herbal medicine |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101410100A (en) * | 2004-01-08 | 2009-04-15 | 黄在宽 | Method and composition for treating acne using lignan compounds |
| WO2006104369A1 (en) * | 2005-03-31 | 2006-10-05 | Amicogen, Inc | Novel use of lignan compounds |
| CN105708926A (en) * | 2016-01-28 | 2016-06-29 | 西南大学 | Compound Chinese herbal medicine for inhibiting and killing aeromonas hydrophila as well as preparation method and application of compound Chinese herbal medicine |
Non-Patent Citations (4)
| Title |
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| CHUNG, J.Y.,ET.AL: ""Anticariogenic Activity of Macelignan Isolated from Myristica Fragrans (Nutmeg) against Streptococcus Mutans"" * |
| RUKAYADI Y.,ET.AL: ""In vitro anti-biofilm activity of macelignan isolated from Myristica fragrans Houtt. against oral primary colonizer bacteria"" * |
| 何秀玲，等: ""抗多种植物病原真菌的肉豆蔻木酚素的分离与抗菌活性研究"" * |
| 张海宾,等: ""12种中草药对嗜水气单胞菌杀伤能力的研究"" * |
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