CN112294796B - Application of ampentaipine in resisting aeromonas hydrophila infection of aquatic animals - Google Patents
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Abstract
The invention discloses application of an amptothecin in resisting infection of water-producing animal aeromonas hydrophila, wherein the amptothecin has no influence on growth of the aeromonas hydrophila, but can reduce pathogenicity of the aeromonas hydrophila by inhibiting expression of the aeromonas hydrophila aerolysin, and further confirms in vivo treatment effect of the amptothecin on the aeromonas hydrophila infection by establishing an animal infection model. The ampentalipin is one of chemical components of Chinese medicine schisandra fruit, has various biological activities and has no toxicity to animals and human beings, so that the ampentalipin can be prepared into pharmaceutical preparations or feed additives for preventing and treating aeromonas hydrophila disease of aquatic animals.
Description
Technical Field
The invention belongs to the technical field of aquaculture, and particularly relates to application of amptothecin in resisting aeromonas hydrophila infection of a water-producing animal.
Background
Fructus Schisandrae chinensis, also called WUMEI, SHANZAO, etc., is a dry fruit of fructus Schisandrae chinensis of Schisandra of Magnoliaceae, and is listed as the superior Chinese medicinal material in Shennong's herbal channels, is a well-known Chinese medicine with warm nature, sweet and sour taste, and has effects of astringing, invigorating qi, promoting fluid production, calming heart and invigorating kidney. Is mainly used for treating lung deficiency, asthma and cough, spontaneous perspiration, night sweat and the like. Modern medicine researches show that schisandra fruit contains various chemical components such as schizandrin, schisandra ester and the like, and the extract has the pharmacological effects of protecting liver, suppressing central nervous system, strengthening heart, reducing blood pressure, enhancing immunity, resisting bacteria and the like; can be used for treating icteric infectious hepatitis, acute intestinal infection, neurasthenia, and bacterial cystitis.
Aeromonas hydrophila is a gram negative bacterium, is widely distributed in natural environment, and is a common bacterium in aquaculture water. The bacterium is a conditional pathogenic bacterium and can infect land animals, aquatic animals and human beings. The aeromonas hydrophila infection of aquatic animals in the freshwater aquaculture often causes explosive hemorrhagic disease, can infect most of the aquatic animals, is a virulent infectious disease of pond culture, and often causes higher mortality and serious economic loss. Chemical antibacterial drugs are the main means of aquaculture for the treatment of bacterial diseases, but due to the unreasonable use of antibacterial drugs, extremely serious drug resistance problems are caused. At present, a large number of drug-resistant aeromonas hydrophila strains insensitive to antibacterial drugs are reported at home and abroad, and great difficulty is brought to the treatment of aeromonas hydrophila diseases of aquatic animals. In addition, in order to control diseases caused by drug-resistant strains, the dosage of the antibacterial drugs is inevitably increased, so that the antibacterial drugs are accumulated and remained in aquatic animal foods, and potential influences are brought to food quality and safety. In conclusion, screening medicines with a brand new anti-infection mechanism has important significance for guaranteeing healthy development of aquaculture industry.
Aerolysin is one of the important virulence factors secreted by aeromonas hydrophila and is a marker for identifying pathogenic aeromonas hydrophila. The research shows that the pathogenicity of aerolysin is obviously reduced after the aerolysin gene is knocked out by aeromonas hydrophila, which shows that the aerolysin plays a key role in the pathogenic process of the aeromonas hydrophila and can be used as a target for developing new drugs.
Disclosure of Invention
The invention aims to provide application of amptothecin in resisting aeromonas hydrophila infection of aquatic animals, and the amptothecin reduces expression of aerolysin by inhibiting transcription of aerolysin coding genes, so that effect of reducing pathogenicity of the amptothecin on the aquatic animals is realized.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the application of the ampentaipine in resisting the infection of aeromonas hydrophila of the aquatic animals: experiments prove that when the concentration of the ampentactin reaches more than 2 mug/mL, the hemolytic activity of the supernatant of the aeromonas hydrophila co-culture can be obviously inhibited, and the transcription of an aerolysin encoding gene aerosa can be obviously inhibited; the channel catfish infected with aeromonas hydrophila is infused with the bupivarin with the dosage of 20mg/kg for 3 days, and the survival rate after treatment can reach more than 60 percent.
The ampentalipin is one of the chemical components of the Chinese medicinal schisandra chinensis, has no toxic or side effect on animals and humans, 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 ampentaipine is not directly sterilized, but reduces the pathogenicity of the ampentaipine, and is not easy to generate drug resistance; the ampentalipin directly acts on the expression of aerolysin, and has therapeutic effect on drug-resistant aeromonas hydrophila; the anwuzhitin is a natural compound, is environment-friendly, is nontoxic to animals, and reduces the risk of drug residues.
Drawings
FIG. 1 is a graph showing the effect of ampentactin on the growth of Aeromonas hydrophila
FIG. 2 is a graph showing the effect of amptothecin on haemolytic activity of Aeromonas hydrophila culture supernatant
FIG. 3 shows the effect of ampentaipine on transcription of aerolysin encoding gene aerolysin A of Aeromonas hydrophila
Detailed Description
Example 1: effect of ampentaipine on Aeromonas hydrophila growth
As in the present embodimentThe related aeromonas hydrophila XS-91-4-1 strain is preserved by aquatic animal pharmacology and medicine residue control technology laboratory of the Yangtze river aquatic institute. XS-91-4-1 strain was cultured in BHI broth until the log phase (OD) 600nm The cultured bacteria were packed in 6 conical flasks of 50mL, 20mL each, and different concentrations (0,1,2,4,8. Mu.g/mL) of ampoules (Sichuan Keqi Biotechnology Co., ltd.) were added to the flask, and the culture was continued at 28℃for 5 hours, with OD measured every 30min 600nm Is a component of the absorption value of (a). FIG. 1 shows the growth curves of different concentrations of amptothecin co-cultured with strain Aeromonas hydrophila XS-91-4-1, from which it can be seen that amptothecin had no effect on Aeromonas hydrophila growth at the concentrations tested.
Example 2: effect of ampentaipine on haemolytic Activity of Aeromonas hydrophila supernatant
Aeromonas hydrophila XS-91-4-1 was cultured overnight in LB liquid medium. The following day is 1:100 was transferred to 200mL of fresh LB liquid medium and the culture was continued until OD 600nm About 0.3, 5 bottles were filled, and ampoules were added to give concentrations 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 OD 600nm When the ratio was 1.5, the bacterial liquid was collected, and the supernatant was centrifuged at 12000rpm for 1min to measure the hemolytic activity of the supernatant. The method comprises the following steps: adding 875 μl of hemolysis buffer (20mM Tris 7.2,0.9%NaCl), 100 μl of bacterial liquid supernatant and 25 μl of defibrinated sheep erythrocytes into 1.5mL centrifuge tube, mixing, standing in 37 deg.C incubator for 15min, centrifuging at 12000rpm, and determining OD of the supernatant 543nm Is a component of the absorption value of (a). As shown in fig. 2, the hemolysis of the supernatant gradually decreased with increasing concentration of ampwulin, and was dose-dependent. Through a hemolysis test, the enwustibin can reduce the hemolytic activity of the supernatant of aeromonas hydrophila, which suggests that the medicine can act on the expression of the aerolysin.
Example 3: effect of Anwu-lipin on transcription of aerolysin-encoding Gene aerosa
Aeromonas hydrophila XS-91-4-1 was cultured in LB liquid medium to a logarithmic growth phase (OD) 600nm =0.3, 28 ℃), and the cultures were placed separately5 conical flasks of 50mL, 20mL of bacterial liquid per flask, were each added with an ampentaplicin concentration of 8,4,2,1,0. Mu.g/mL, and the culture was continued at 28℃until OD 600nm The cells were collected by high speed centrifugation (8000 g,1 min) to 1.5. The thallus adopts a total RNA extraction kit of the root bacteria to extract thallus RNA, adopts a Takara RNA PCR kit (AMV) kit to synthesize cDNA, uses a SYBR Premix Ex Taq kit to carry out a fluorescent quantitative PCR test, analyzes the expression level of an aerolysin coding gene aerosil by a delta Ct method, and uses 16s rRNA as an internal reference. 3 independent replicates were run for each drug concentration group. The primers used in the test were synthesized by Shanghai Biotechnology and the sequences are as follows: upstream primers for aero gene (aero-F): 5'-TCTACCACCACCTCCCTGTC-3', downstream primer (aero-R): 5'-GACGAAGGTGTGGTTCCAGT-3' the molecular weight of the amplified product is 218bp.16s rRNA upstream primer (16 s rRNA-F): 5' -TAATACCGCATACGCCCTAC-3, downstream primer (16 s rRNA-R): 5'-ACCGTGTCTCAGTTCCAGTG-3' the molecular weight of the amplified product is 164bp. As shown in FIG. 3, the transcription of the aerolysin-encoding gene aeroA can be significantly inhibited after the concentration of the ampwustiin reaches more than 2 mug/mL, and the inhibition effect is dose-dependent. This result suggests that ampwustiin reduces the amount of expression of aerolysin in culture supernatant by inhibiting transcription of aaera.
Example 4: therapeutic effect of ampentaipine on aeromonas hydrophila infection model of channel catfish
60 healthy channel catfish (from the clinical test center of fishery medicine of the Yangtze river institute) are respectively placed in 3 200L glass cylinders, 20 channels are respectively placed in each group, the water temperature is kept at 28 ℃, the dissolved oxygen is 5.5-5.7mg/L, and 7d is temporarily raised before toxicity attack. Aeromonas hydrophila XS-91-4-1 strain was cultured in LB medium to mid-log growth (OD) 600nm =1.0), the bacterial solution was centrifuged, and the bacterial cells were washed 2 times with sterile PBS and resuspended to 1.5×10 with a mahogany tube 8 CFU/mL, the positive control group and the treatment group were each injected intraperitoneally with 200. Mu.L of diluted bacterial solution, and the negative control group was intraperitoneally with 200. Mu.L of sterile PBS. The treatment groups were orally infused with 20mg/kg of amptotipol 6h post-infection, once every 12h, for three consecutive days, and the death of each group of fish was observed and recorded daily. As shown in Table 1, the channel catfish infected with Aeromonas hydrophila was not administeredThe mortality rate of the drug treatment is 90% in 5d, and the mortality rate of the drug treatment after 20mg/kg of ampwustiin treatment is 35% and is lower than that of the group without infection. The results show that the ampentaipine can be used for treating the aeromonas hydrophila infection of the channel catfish, and the dosage is 20mg/kg, and the ampentaipine is administrated once every 12 hours for 3 days.
TABLE 1 therapeutic Effect of ampentaipine on Aeromonas hydrophila infection model of Ictalurus punctatus
The small knot:
it was found from example 1 that amptothecin had no effect on the growth of Aeromonas hydrophila XS-91-4-1 strain at the tested concentration, suggesting that amptothecin had no antibacterial effect on Aeromonas hydrophila at and below this concentration. It was found by example 2 that enwustippling was able to reduce the hemolytic activity of aeromonas hydrophila culture supernatant in a dose-dependent manner, suggesting that enwustippling inhibits the expression or activity of aerolysin. Further, it was found from example 3 that ampwustiin can inhibit transcription of aerolysin-encoding gene aerosa, thereby reducing the expression level of aerolysin in the supernatant. Finally, the study of the example 4 shows that the 20mg/kg amptothecin treatment can reduce the death rate of the aeromonas hydrophila model of the channel catfish, and the amptothecin can be used as a medicament for treating the aeromonas hydrophila infection of the channel catfish.
Aeromonas hydrophila is a conditional pathogen widely existing in aquaculture environments, and can cause the morbidity of most freshwater aquaculture animals, such as silver carp, grass carp, chinese soft-shelled turtle, procambarus clarkii and the like. Aerolysin is the material basis for aeromonas hydrophila to cause various infections, and is prevalent in pathogenic aeromonas hydrophila strains. From this, it can be inferred that amptothecin has therapeutic effects on aeromonas hydrophila infection in aquaculture animals.
Claims (2)
1. Application of anwuzhein in preparing medicine for preventing and treating aeromonas hydrophila infection of aquatic animal is provided.
2. The use according to claim 1, wherein the amount of ampwustine administered is greater than 20 mg/kg.
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