CN114869871A - Application of pterostilbene in resisting infection of aquatic aeromonas hydrophila of aquaculture animals - Google Patents
Application of pterostilbene in resisting infection of aquatic aeromonas hydrophila of aquaculture animals Download PDFInfo
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- CN114869871A CN114869871A CN202210611913.1A CN202210611913A CN114869871A CN 114869871 A CN114869871 A CN 114869871A CN 202210611913 A CN202210611913 A CN 202210611913A CN 114869871 A CN114869871 A CN 114869871A
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Abstract
The invention discloses application of pterostilbene in resisting aquatic animal aeromonas hydrophila infection, wherein the pterostilbene has no inhibition effect on the growth of aeromonas hydrophila in vitro, but can directly inhibit the activity of aerolysin, biofilm formation and quorum sensing, so that the in-vivo and in-vitro pathogenicity of the aeromonas hydrophila is reduced, and the survival rate of a channel catfish infection model is improved. The pterostilbene mainly comes from pterocarpus indicus, blueberries, grapes and the like, has the effects of oxidation resistance, inflammation resistance, fungus resistance and the like, is high in safety to animals and people, and can be used as a medicine or feed additive for preventing and treating the aeromonas hydrophila disease of aquaculture animals.
Description
Technical Field
The invention belongs to the technical field of aquaculture, and particularly relates to application of pterostilbene in resisting infection of aquatic aeromonas hydrophila of aquaculture animals.
Background
Aeromonas hydrophila is one of the main pathogenic bacteria seriously threatening the healthy development of the freshwater aquaculture industry, is a malignant disease of pond culture, often causes diseases such as bacterial septicemia of cultured fishes and the like, and is widely distributed in the aquaculture environment. Aeromonas hydrophila is a pathogenic bacterium of zoonosis and fish, which can cause various infections of terrestrial animals and humans. Furthermore, Aeromonas hydrophila is also considered to be a food-borne pathogen because it can infect humans through contaminated water, uncooked aquatic products, and the like. Aeromonas hydrophila disease in aquaculture mainly depends on antibiotics for treatment, such as enrofloxacin, florfenicol and the like. But the occurrence of bacterial drug resistance due to the improper use of the drug leads to the reduction of the treatment effect and even the failure of the treatment. In addition, the use of antibiotics accumulates in the fish body, and has potential threats to the quality safety of aquatic products and human health. Therefore, the screening of natural compounds for drugs that directly inhibit the pathogenicity of Aeromonas hydrophila has become a focus of research in recent years, and the screened drugs are also called anti-toxic drugs. The anti-virulence drugs mainly aim at virulence factors of pathogenic bacteria and cannot generate selective pressure on the survival of the pathogenic bacteria, so that drug resistance is not easy to generate. Aerolysin is a main virulence factor of aeromonas hydrophila and is closely related to the pathogenicity of aeromonas hydrophila, so that the aerolysin becomes a target for researching anti-aeromonas hydrophila infection medicines.
The pterocarpus santalinus is the heartwood of the pterocarpus santalinus of the leguminosae and is distributed in Fujian, Guangdong and other places in China. The red sandalwood is salty in taste and neutral in nature, enters liver meridian, has the effects of removing blood stasis, regulating nutrient, stopping bleeding, relieving pain, detoxifying and reducing swelling, and is mainly used for treating headache, cardiac and abdominal pain, incomplete lochiorrhea, pain in urination, wind-toxin carbuncle swelling, incised wound bleeding and other diseases. Pterostilbene is one of the main chemical components of pterocarpus indicus, also called pterostilbene, and has biological activities of resisting oxidation, resisting cancer, resisting inflammation, resisting fungi and the like.
Disclosure of Invention
The invention aims to provide application of pterostilbene in resisting aeromonas hydrophila infection of aquaculture animals, wherein the pterostilbene inhibits in vivo and in vitro pathogenicity of aeromonas hydrophila by directly inhibiting aerosol activity, biofilm formation and quorum sensing, but has no influence on growth of the aeromonas hydrophila.
In order to achieve the purpose, the invention adopts the following technical scheme:
the application of pterostilbene in preparing the medicament or the additive for resisting the infection of the aeromonas hydrophila of the aquaculture animals comprises the following steps: experiments prove that the pterostilbene has no inhibition effect on the growth of aeromonas hydrophila between 0 and 16 mu g/mL; but when the concentration of the pterostilbene reaches 2 mug/mL or more, the expression of the aerolysin and the formation of the biofilm can be obviously inhibited; when the concentration of the pterostilbene is 2 mug/mL or more, the activity of the purified aerolysin can be obviously inhibited; through a fluorescent quantitative PCR test, the pterostilbene can obviously inhibit the transcription of quorum sensing related genes ahyI and ahyR and an aerosol encoding gene aerA when the concentration of the pterostilbene is 16 mu g/mL; oligomerization tests show that the pterostilbene can inhibit the formation of an aerolysin heptamer and further inhibit the activity of the aerolysin heptamer; through establishing the model of the channel catfish aeromonas hydrophila, the survival rate of the channel catfish aeromonas hydrophila infection model can be obviously improved by pouring 25mg/kg of pterostilbene into the mouth.
The pterostilbene is mainly extracted from traditional Chinese medicine pterocarpus indicus or blueberries, grapes and the like in fruits, has high safety to animals and people, and can be used as a medicine or feed additive for preventing and treating aeromonas hydrophila infection of aquaculture animals.
Compared with the prior art, the invention has the following advantages and effects: the pterostilbene is a natural compound derived from plants, has no toxic or side effect, is environment-friendly, can reduce the use of chemical antibacterial agents in aquaculture, and is beneficial to reducing the residues of the chemical antibacterial agents in the aquaculture and the influence of the chemical antibacterial agents on environmental microorganisms. In addition, the pterostilbene only reduces the toxicity of aeromonas hydrophila, but has no obvious inhibition effect on the growth of the aeromonas hydrophila, so that the selective pressure caused by the pterostilbene is low, and the drug resistance is not easy to generate.
Drawings
FIG. 1 is a graph of the effect of pterostilbene on the growth of Aeromonas hydrophila.
FIG. 2A is a graph of the effect of pterostilbene on hemolytic activity of Aeromonas hydrophila culture supernatant.
FIG. 2B shows the inhibitory effect of pterostilbene on the hemolytic activity of purified aerolysin.
FIG. 3 is a graph of the effect of pterostilbene on Aeromonas hydrophila biofilm formation.
FIG. 4 is a graph of the effect of pterostilbene on the aerolysin content of the supernatant of an Aeromonas hydrophila culture.
FIG. 5 is a graph of the effect of pterostilbene on expression of related genes.
FIG. 6 is a graph of inhibition of pterostilbene on the formation of an aerolysin heptamer.
Detailed Description
Example 1: influence of pterostilbene on the growth of Aeromonas hydrophila.
The aeromonas hydrophila 168 strain is preserved in a laboratory of aquatic animal pharmacology and drug residue control technology of Yangtze river aquatic research institute. 168 strains were cultured in LB liquid medium to the logarithmic growth phase (OD) 600nm 0.3, 28 ℃), subpackaging the cultured bacteria into 6 conical flasks with 50mL, adding pterostilbene (WKQ-0039373) with different concentrations (0, 2, 4, 8, 16 and 32 mu g/mL) into each flask with 20mL, culturing for 5h at 28 ℃, taking the bacteria liquid every 30min, and measuring OD (optical density) once 600nm The absorption value of (2). FIG. 1 shows the growth curve of co-culture of pterostilbene and Aeromonas hydrophila 168 strain at different concentrations, and it can be seen from the figure that pterostilbene has no effect on the growth of Aeromonas hydrophila at a concentration of 2-16. mu.g/mL, but can inhibit the growth of Aeromonas hydrophila at 32. mu.g/mL.
Example 2: influence of pterostilbene on hemolytic activity of Aeromonas hydrophila supernatant
The aeromonas hydrophila 168 strain is preserved by the research institute of aquatic products in Yangtze river. Aeromonas hydrophila 168 was cultured overnight in LB liquid medium. The following day is as follows: 100 volume ratio was transferred to 200mL of fresh LB liquid medium, and the culture was continued to OD 600nm When the concentration is about 0.3, the mixture is divided into 5 bottles, and pterostilbene is added to make the concentration reach 0, 2, 4, 8 and 16 mu g/mL. Continuing to culture until OD 600nm The bacterial solution was collected at 1.5, centrifuged at 12000rpm for 1min and the supernatant was assayed for hemolytic activity. As shown in FIG. 2A, as the concentration of pterostilbene increasedThe hemolysis of the supernatant was gradually reduced and dose-dependent. When the concentration of the pterostilbene is 2 mu g/mL or more, the hemolytic activity of the supernatant of the aeromonas hydrophila culture can be remarkably inhibited.
Example 3: inhibition of purified aerolysin hemolytic activity by pterostilbene
The aerosol essence is prepared and stored in aquatic animal pharmacology and drug residue control technology laboratory of Yangtze river aquatic research institute. Adding 975 μ L of hemolysis buffer solution and 2 μ L of purified aerosil into 1.5mL centrifuge tube, adding different amount of pterostilbene mother liquor to make final concentration of 2, 4, 8, 16 μ g/mL, setting negative control without drug, reacting in 37 deg.C incubator for 15min, adding 25 μ L (5 × 10) per tube 6 One per mL) of defibered sheep red blood cells, and placing the defibered sheep red blood cells into a constant temperature incubator at 37 ℃ for reaction for 15 min. After the reaction was complete, the reaction mixture was centrifuged at 12000 Xg for 1min, and the supernatant was collected at OD 543nm The absorption value is measured. The group to which 0.1% Triton X-100 was added was used as a positive control (100% hemolytic group). As shown in fig. 2B, pterostilbene can decrease the hemolytic activity of purified aerolysin dose-dependently, and when the pterostilbene concentration is 2 μ g/mL or more, the hemolytic activity of aerolysin can be significantly inhibited.
Example 4: inhibition effect of pterostilbene on aeromonas hydrophila biofilm formation
A single colony of Aeromonas hydrophila 168 is selected and inoculated in a BHI liquid culture medium, after shaking culture is carried out at 28 ℃ and 180rpm until OD600nm is 1.0, the bacterial liquid is diluted by 10 times by using the BHI (HB 8297-5, Qingdao Haibo biotechnology limited) liquid culture medium containing 2% of glucose, the diluted bacterial liquid is added into a 96-well plate, 200 mu L of each well reaction system is adopted, the final concentration of pterostilbene is 2, 4, 8 and 16 mu g/mL, and a negative control group only containing the culture medium and a positive control group containing the culture medium and the bacterial liquid are arranged. Culturing in an incubator at 28 ℃ for 24h at constant temperature, taking out, discarding liquid, washing with PBS for 3 times, putting into an oven, drying, adding 200 mu L of 0.5% crystal violet into each hole, reacting at 37 ℃ for 0.5h, discarding liquid, washing off redundant crystal violet, drying, adding 30% glacial acetic acid into each hole for 200 mu L, after the bottom solid is dissolved, measuring the light absorption value at 570nm with an enzyme labeling instrument, and repeating each group of tests for 3 times. As shown in fig. 3, pterostilbene can dose-dependently reduce the formation of aeromonas hydrophila biofilms.
Example 5: influence of pterostilbene on the content of aerolysin in supernatant of Aeromonas hydrophila culture
20 mu L of the supernatant obtained in example 2 was added with an appropriate amount of sample preparation buffer, and then boiled in boiling water for 8min to prepare a sample, which was then taken out and centrifuged at high speed for 1 min. A10. mu.L sample was added to SDS-PAGE gel and subjected to electrophoresis. And (4) taking out the protein gel after the electrophoresis is finished, and transferring the protein to the PVDF membrane by a semi-dry transfer printing instrument. The unbound membrane sites were blocked with 5% skim milk powder, then an anti-aerolysin polyclonal antibody (stored in the laboratory) was added and incubated for 1h, PBS was washed 3 times, and a horseradish peroxidase-labeled secondary antibody (Wuhanding national bio-corporation) was added, and after incubation for 1h, washing was continued for 3 times, and after adding ECL luminescence solution and reacting for 3min, the target band was detected on a chemiluminescence apparatus. As shown in fig. 4, pterostilbene can reduce the content of aerolysin in the supernatant dose-dependently, and in combination with examples 2 and 4, it is speculated that pterostilbene may reduce aerosol expression and biofilm formation by suppressing quorum sensing.
Example 6: influence of pterostilbene on transcription of aerA, ahyI and ahyR genes
The aeromonas hydrophila 168 strain is preserved by the research institute of aquatic products in Yangtze river. Aeromonas hydrophila 168 strain is cultured in LB liquid medium to logarithmic growth prophase (OD) 600nm 0.3, 28 ℃), placing the culture bacteria into 2 conical flasks of 50mL, respectively, adding pterostilbene with the concentration of 16 and 0 mu g/mL into each 20mL bacterial liquid, and continuously culturing at 28 ℃ until the OD is reached 600nm The cells were collected by high speed centrifugation (8000 Xg, 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 fluorescence quantitative PCR test, analyzes the expression levels of aerA, ahyI and ahyR genes by a delta Ct method, and takes 16s rRNA as an internal reference. The primers used in the experiments were synthesized by Shanghai Biotech and the sequences are shown in Table 1. As shown in FIG. 5, the genes aerA, ahyI and ahyR were down-regulated by 8.54, 10.1 and 4.98 times, respectively, when the pterostilbene concentration was 16. mu.g/mL. The results suggest that pterostilbene can inhibit quorum sensingReduce biofilm formation and expression of aerolysin.
TABLE 1 primer sequences for fluorescent quantitative PCR
Example 7: influence of pterostilbene on formation of aerolysin heptamer
Mixing the activated aerolysin and pterostilbene at the same molar ratio in an active hemolysis test, reacting at room temperature for 15min, adding Hepes to induce oligomerization test, standing at room temperature for 1h, adding loading buffer, performing water bath at 100 ℃ for 10min, and performing gel electrophoresis test by 8% SDS-PAGE. Adding a dyeing solution, shaking for 2h for dyeing, adding a decolorizing solution, shaking for 2-3h on a shaking table, washing, and taking a picture. As shown in fig. 6, as the concentration of pterostilbene increased, the heptamer gradually decreased, indicating that pterostilbene decreased its hemolytic activity by inhibiting the formation of the aerolysin heptamer.
Example 8: therapeutic effect of pterostilbene on model infected with aeromonas hydrophila of channel catfish
The aeromonas hydrophila 168 strain is preserved by the research institute of aquatic products in Yangtze river. The channel catfish is from the fishing medicine clinical test center of the Changjiang aquatic research institute. The 90 healthy channel catfish is respectively placed in 3 200L glass jars, 20 tails of each group are kept at the water temperature of 28 ℃, the dissolved oxygen is 5.5-5.7mg/L, and the channel catfish is temporarily cultured for 7 days before toxicity attack. Aeromonas hydrophila 168 strain is cultured in LB culture medium to logarithmic growth phase (OD) 600nm 1.0), the bacterial solution was centrifuged, the cells were washed 2 times with sterile PBS and resuspended to 1.5 × 10 using a turbidimetric tube 8 CFU/mL, and 200 mu L of diluted bacterial liquid is injected into the abdominal cavity of each fish in the positive control group and the treatment group, and 200 mu L of sterile PBS is injected into the abdominal cavity of the negative control group. The treatment group is administered with 25mg/kg pterostilbene 6h after infection, once every 12h, continuously for three days, and observed and recorded every dayMortality of the herds. As shown in Table 2, the mortality rate of the channel catfish infected with Aeromonas hydrophila within 8d without drug treatment is 90%, the mortality rate after 25mg/kg of pterostilbene treatment is 43.43%, and no death occurs during the negative control group test. The results show that the pterostilbene can be used for treating the aeromonas hydrophila infection of the channel catfish, the dosage of the pterostilbene is 25mg/kg, the pterostilbene is administrated once every 12 hours, and the pterostilbene is continuously used for 3 days.
TABLE 2 mortality following treatment of the model for Aeromonas hydrophila infection of Ictalurus punctatus by pterostilbene
The embodiment 1 finds that the pterostilbene has no influence on the growth of the aeromonas hydrophila at the concentration of 2-16 mu g/mL, and the suggestion is that the pterostilbene does not generate selective pressure on the aeromonas hydrophila in the application process, and different from the traditional antibiotics, the pterostilbene does not induce pathogenic bacteria to generate drug resistance. Further, it is found from examples 2 and 3 that pterostilbene has an inhibitory effect on hemolytic activity of the supernatant co-cultured with Aeromonas hydrophila and purified aerolysin and is dose-dependent, suggesting that pterostilbene can inhibit activity or expression of aerolysin. Through example 5, the pterostilbene has an inhibiting effect on the content of aerolysin in the supernatant of the co-culture, and a conclusion is preliminarily drawn by combining examples 2 and 3 that the pterostilbene can inhibit the expression of the aerolysin and directly reduce the activity of the aerolysin. The pterostilbene is found to have an inhibiting effect on biofilm in example 4, and the combination of example 2 suggests that the drug may act on quorum sensing, and the confirmation is carried out in example 6. Furthermore, it was found by example 7 that pterostilbene can be rendered inactive by inhibiting the formation of functional heptamers by aerosols. Since both the aerolysin and the biofilm are closely related to the pathogenicity of the aeromonas hydrophila, the examples show that the pterostilbene can reduce the in-vivo pathogenicity of the aeromonas hydrophila by inhibiting virulence, and the example 8 establishes the model for the infection of the aeromonas hydrophila by the channel catfish, finds that the 25mg/kg pterostilbene can obviously reduce the death rate of the channel catfish infected with the aeromonas hydrophila by the channel catfish, and shows that the pterostilbene can be used as a medicament or an additive for preventing or treating the aeromonas hydrophila infection.
Aeromonas hydrophila is a common pathogenic bacterium in freshwater aquaculture and can infect most freshwater aquaculture animals, such as crayfish, crucian, pseudobagrus fulvidraco and the like. The existing research finds that the aerolysin is the main virulence factor of the aeromonas hydrophila, the purpose of controlling the pathogenicity of the aeromonas hydrophila can be achieved by inhibiting the activity or regulating and controlling, and the method is generally suitable for the aeromonas hydrophila from different sources. Therefore, the pterostilbene has prevention or treatment effect on the aeromonas hydrophila infection of the aquaculture animals.
Claims (3)
1. Application of pterostilbene in preparing medicine for preventing or treating aeromonas hydrophila infection of aquaculture animals.
2. Application of pterostilbene in preparing additive for preventing and treating aeromonas hydrophila infection of aquaculture animals.
3. The use as claimed in claim 1, wherein the pterostilbene is administered at a dose of greater than 25 mg/kg.
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Citations (2)
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| CN102696592A (en) * | 2012-04-20 | 2012-10-03 | 中国人民解放军第二军医大学 | New application of pterostilbene to anti-fungal biofilm |
| CN109044995A (en) * | 2018-07-20 | 2018-12-21 | 中国水产科学研究院淡水渔业研究中心 | It is a kind of for preventing and treating the drug of bacillary hemorrhage caused by Aeromonas hydrophila |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102696592A (en) * | 2012-04-20 | 2012-10-03 | 中国人民解放军第二军医大学 | New application of pterostilbene to anti-fungal biofilm |
| CN109044995A (en) * | 2018-07-20 | 2018-12-21 | 中国水产科学研究院淡水渔业研究中心 | It is a kind of for preventing and treating the drug of bacillary hemorrhage caused by Aeromonas hydrophila |
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| PU-YING QI等: "Exploring an Innovative Strategy for Suppressing Bacterial Plant Disease: Excavated Novel Isopropanolamine-Tailored Pterostilbene Derivatives as Potential Antibiofilm Agents", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》, vol. 70, no. 16, pages 4899 - 4911 * |
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