CN116139126A - Application of epigallocatechin-3-gallate in preparing quorum sensing inhibitor of aeromonas hydrophila - Google Patents
Application of epigallocatechin-3-gallate in preparing quorum sensing inhibitor of aeromonas hydrophila Download PDFInfo
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- CN116139126A CN116139126A CN202310368703.9A CN202310368703A CN116139126A CN 116139126 A CN116139126 A CN 116139126A CN 202310368703 A CN202310368703 A CN 202310368703A CN 116139126 A CN116139126 A CN 116139126A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The application is applicable to the technical field of biology and provides application of epigallocatechin-3-gallate in preparing quorum sensing inhibitor of aeromonas hydrophila. According to research on the anti-QS potential of epigallocatechin-3-gallate on aeromonas hydrophila and the protection effect of the epigallocatechin-3-gallate on the aeromonas hydrophila after the aeromonas hydrophila is infected by zebra fish, the research result shows that EGCG inhibits the virulence factors of aeromonas hydrophila biomembrane, hemolysin, swimming and surging and reduces the generation of AI-2 signal molecules; the qRT-PCR result shows that EGCG can inhibit the expression level of QS and virulence factor related genes; animal experiments show that EGCG can remarkably improve the survival rate of the zebra fish infected with aeromonas hydrophila, reduce bacterial load and improve the damage of liver, muscle and gill; EGCG is therefore a very promising quorum sensing inhibitor for alleviating aeromonas hydrophila infection.
Description
Technical Field
The application belongs to the technical field of biology, and particularly relates to application of epigallocatechin-3-gallate in preparing quorum sensing inhibitor of aeromonas hydrophila.
Background
Tea drinking has a long history in China and is an indispensable part of daily life of people. Epigallocatechin-3-gallate (EGCG) is the main active ingredient of green tea, accounting for 59% of total catechin in green tea. Aeromonas hydrophila is a common opportunistic gram-negative zoonotic pathogen that is widely found in various aquatic environments. Aeromonas hydrophila often causes hemorrhagic septicemia in farmed and wild fish, manifested by internal and external bleeding, tail rot, sudden eye death and post-infection death. Infection of humans with aeromonas hydrophila can cause gastroenteritis, hemolytic uremic syndrome, peritonitis, skin infection, meningitis, necrotizing fasciitis, and sepsis in immunocompromised patients. Antibiotics and chemicals are commonly used to control aeromonas hydrophila infection. It often leads to the appearance of "superbacteria" and the accumulation of chemicals in the food chain.
Quorum Sensing (QS) is a density-dependent cell-cell signal that triggers a change in behavior when the population reaches a critical density, dependent on accumulation of an auto-induction factor (AI). When the AI reaches a threshold concentration, a signal cascade is triggered, which promotes the synchronous expression of genes in bacterial populations, such as bioluminescence, virulence factor secretion, biofilm formation, and other biological behaviors. Due to the important role of quorum sensing inhibitors (quorum-sensi ng inhibitors, QSIs) in bacterial pathogenesis, quorum sensing inhibitors of natural origin (quorum-sensing inhibitors, QSIs) have received extensive attention, either alone or in combination with antibiotics, to inhibit bacterial infection and disease progression. To date, there has been little research in the prior art on quorum sensing inhibitors of aeromonas hydrophila, and no application of EGCG to research on the effects of aeromonas hydrophila qs regulatory virulence factors has been seen.
Disclosure of Invention
An aim of the embodiment of the application is to provide an application of epigallocatechin-3-gallate in preparing quorum sensing inhibitor of aeromonas hydrophila.
The embodiment of the application is realized in such a way that the epigallocatechin-3-gallate is applied to the preparation of the quorum sensing inhibitor of aeromonas hydrophila.
Another object of an embodiment of the present application is the use of gallocatechin-3-gallate for the preparation of an active formulation of a virulence factor that inhibits aeromonas hydrophila biofilm, hemolysin, motility and gushing.
Another object of an embodiment of the present application is the use of epigallocatechin-3-gallate for inhibiting quorum sensing-related gene expression and virulence factor-related gene expression in aeromonas hydrophila.
Another object of an embodiment of the present application is the use of epigallocatechin-3-gallate for the manufacture of a medicament for treating or preventing an infection of an organism with aeromonas hydrophila.
Another object of an embodiment of the present application is a quorum sensing inhibitor of aeromonas hydrophila, the active ingredient of the quorum sensing inhibitor comprising epigallocatechin-3-gallate.
Another object of an embodiment of the present application is a medicament for treating or preventing infection of an organism with aeromonas hydrophila, the active ingredient of the medicament comprising epigallocatechin-3-gallate.
According to the embodiment of the application, research on the QS potential of epigallocatechin-3-gallate on aeromonas hydrophila and the protection effect of the epigallocatechin-3-gallate on aeromonas hydrophila after zebra fish is infected with the same shows that EGCG inhibits the virulence factors of aeromonas hydrophila biomembrane, hemolysin, swimming and surging and reduces the generation of AI-2 signal molecules; the qRT-PCR result shows that EGCG can inhibit the expression level of QS and virulence factor related genes; animal experiments show that EGCG can remarkably improve the survival rate of the zebra fish infected with aeromonas hydrophila, reduce bacterial load and improve the damage of liver, muscle and gill; EGCG is therefore a very promising quorum sensing inhibitor for alleviating aeromonas hydrophila infection.
Drawings
FIG. 1 is a graph showing the growth of Aeromonas hydrophila provided in the examples of the present application.
Fig. 2 is a schematic representation of the effect of 24h and 48h EGCG on biofilm formation provided in the examples of the present application.
Fig. 3 is a schematic diagram showing the effect of EGCG provided in the examples of the present application on aeromonas hydrophila surge and swimming motion.
Fig. 4 is a schematic diagram showing the effect of EGCG provided in the examples of the present application on haemolytic activity of aeromonas hydrophila.
FIG. 5 is a schematic diagram showing the results of determining AI-2 activity by the bioluminescence assay provided in the examples herein.
FIG. 6 is a schematic diagram showing the effect of EGCG on Aeromonas hydrophila virulence factor and QS gene expression according to the embodiment of the present application.
Fig. 7 is a schematic diagram showing the effect of EGCG provided in the examples of the present application on the protection effect of zebra fish infection with aeromonas hydrophila.
Fig. 8 is a graph showing bacterial load results of zebra fish after EGCG treatment provided in the examples of the present application.
Fig. 9 is a schematic diagram showing the effect of EGCG provided in the examples of the present application on zebra fish intestinal cytokines.
FIG. 10 is a schematic representation of the results of histopathological analysis of different groups of tissue selected in accordance with the examples provided herein.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present application more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The following example studies were performed on the anti-QS potential of EGCG against aeromonas hydrophila and its protective effect against zebra fish infection after aeromonas hydrophila:
among these, the following examples are investigated for the strains, growth conditions and chemicals used:
clinically isolated aeromonas hydrophila AH08 was provided by the professor Chen Defang (university of agriculture, sichuan) and was used for whole in vitro and in vivo studies. The test strain was routinely cultured in Trypticase Soy Broth (TSB) at 30℃at 150rpm. Strains v.harveyi BB170 and v.harveyi BB152 were provided by Han Xiangan teachings and cultured in AB medium at 30 ℃. All bacterial media were purchased from the maritime organism (peninsula china). EGCG was obtained from Shanghai Seiya Biotechnology Co., ltd (Shanghai, china) and was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution at a concentration of 80 mg/mL. The strain species used are only examples, and should not limit the application scope of the application.
Example 1 influence of EGCG on the anti-QS potential of Aeromonas hydrophila
(1) Effect of EGCG on Aeromonas hydrophila growth
MIC and MBC determination: MIC and MBC of EGCG against aeromonas hydrophila were determined on 96-well plates. The medicine is diluted by a TSB culture medium double method, and EGCG with the concentration of 4096 mug/mL-32 mug/mL is obtained. Incubate at 30℃for 24 hours. The lowest concentration of EGCG that inhibits the visible growth of bacteria is considered the MIC. To determine the Minimum Bactericidal Concentration (MBC) of EGCG on the strain, 100 μl was sampled from wells without bacterial growth and inoculated onto TSB agar. Bacterial growth was monitored to determine MBC by incubation at 30 ℃ for 24 hours.
Growth curve: the effect of EGCG on bacterial growth was determined by the growth curve method. Aeromonas hydrophila was grown overnight and then the bacterial culture was diluted with fresh TSB medium (50 mL) to achieve CFU of 5 x 105. EGCG (0-256. Mu.g/mL) at different concentrations was incubated with aeromonas hydrophila cultures at 30℃with shaking (150 rpm). Culture suspension without test compound served as control. The od600nm value was recorded every 1h with a spectrophotometer and the effect of EGCG on Aeromonas hydrophila was monitored.
Experimental results: the growth curve of aeromonas hydrophila is shown in fig. 1, wherein all data in the graph represent three independent experiments, repeated three times each time, expressed as mean ± SD of each histogram. EGCG has a MIC of 1024. Mu.g/ml for hydrophila and MBC of 4096. Mu.g/ml. Subsequent experiments were performed at sub-mic concentrations (256, 128, 64, 32 and 16. Mu.g/ml). The growth curve shows that EGCG increases the lag phase of the bacteria, but after 12h there is no significant inhibition of bacterial growth.
(2) Effect of EGCG on biofilm formation
Biofilm inhibition assay: EGCG (0, 16, 32, 64, 128 and 256. Mu.g/mL) was incubated at 30℃in 96-well plates for 24h or 48h, respectively. After incubation, the cultures were removed and the dishes were carefully washed three times with PBS. Then 200 μl of 0.5% crystal violet was transferred to the well to stain the biofilm. The free crystal violet was rinsed with water. The plate was dried and 200. Mu.L of 33% glacial acetic acid was used to dissolve the crystal violet-bound biofilm. The absorbance was measured at 570 nm.
Experimental results: the effect of 24h and 48h EGCG on biofilm formation as shown in fig. 2, wherein all data in the graph represent three independent experiments, repeated three times each, expressed as mean ± SD of each histogram, p <0.05; * P <0.01; * P <0.001.EGCG significantly inhibited the formation of aeromonas hydrophila biofilm. When EGCG concentration was 128. Mu.g/ml, the inhibition rate against biofilm was 38.27% at 24h, 26.62% at 48h, and EGCG inhibition against biofilm was concentration-dependent at 24 h.
(3) Inhibiting clustering and swimming movements
Motility assay: in swimming experiments, overnight-cultured hydrophile were carefully inoculated onto swimming agar plates (agar 0.3%, peptone 1%, glucose 1% and NaCl 0.5%) with or without EGCG added, respectively (256, 128 and 64. Mu.g/mL). After incubation for 18h at 30 ℃, the diffuse colony area was determined. With or without EGCG, 6. Mu.L of bacterial suspension was transferred to the center of the prepared colony agar plates (agar 0.5%, peptone 1%, naCl 0.5%, glucose 0.5%). Migration zone was recorded after incubation at 30 ℃ for 18 h.
Experimental results: effect of EGCG on aeromonas hydrophila surge and swimming movement as shown in fig. 3: (A) gushing and swimming, (B) aeromonas hydrophila gushing movement distance diameter, (C) aeromonas hydrophila swimming movement distance. All data in the figures represent three independent experiments, repeated three times each, expressed as mean ± SD of each histogram. * p <0.05; * P <0.01; * P <0.001.EGCG significantly inhibited the motility of aeromonas hydrophila and was concentration dependent. Under the action of surging, the inhibition rates of 128 mug/mL and 256 mug/mL EGCG on the water fleas are 29.72 percent and 32.97 percent respectively. The inhibition rates of EGCG during swimming were 16.03%, 28.30% and 66.03% for 64. Mu.g/mL, 128. Mu.g/mL and 256. Mu.g/mL, respectively. The motility of aeromonas hydrophila is associated with flagella, and flagella-driven motility plays an important role in the pathogenicity of bacteria. The play and cluster are regulated by the QS system.
(4) Inhibiting hemolysin
Quantitative determination of beta-hemolysin: extracellular hemolysin production by aeromonas hydrophila was quantified according to a slightly modified study from the previous. Briefly, 100. Mu.l each of treated and untreated Aeromonas hydrophila was incubated with 900. Mu.l phosphate buffered saline containing 5% rabbit erythrocytes, followed by incubation at 4℃for 1 hour. The mixture was centrifuged and the supernatant was collected and measured at 530 nm.
Experimental results: the effect of EGCG on haemolytic activity of Aeromonas hydrophila as shown in FIG. 4. All data in the figures represent three independent experiments, repeated three times each, expressed as mean ± SD of each histogram. * p <0.05, p <0.01. High concentrations of EGCG significantly inhibited the hemolytic activity of aeromonas hydrophila. Compared with the blank control, the inhibition rate of 256 mug/ml EGCG to hemolysin is 28.2 percent.
(5) Influence of EGCG on AI-2 production
AI-2 detection: the hydrophillic bacteria were cultured overnight with varying concentrations of EGCG (0, 16, 32, 64, 128 and 256. Mu.g/mL). Bacteria culture was centrifuged at 8000g for 5min, and the supernatant was filtered through a 0.22 μm filter to collect cell-free culture broth. Report strain v.harveyi BB152 was cultured overnight in AB medium at 30 ℃ with continuous shaking.
Strain v.harveyi BB170 was cultured in AB medium at 30 ℃ for 18h and then diluted 1:2000 into fresh AB medium. 180. Mu.L of diluted cells were added to wells of a 96-well plate and mixed with 20. Mu.L of cell-free supernatant, hydrophila A.hydrophila or V.harveyi BB 152. AI-2 bioassays were performed three times for each sample. The experiment was repeated three times. AI-2 activity was quantified from AI-2 relative concentration as follows:
where Nt is the bioluminescence of the test sample and Np is the bioluminescence of the positive control.
Experimental results: AI-2 activity was measured by bioluminescence as shown in FIG. 5. All data in the figures represent three independent experiments, repeated three times each, expressed as mean ± SD of each histogram. * p <0.05; * P <0.01; * P <0.001. The AI-2 secretion by the hydrophillic bacteria was significantly reduced in the presence of EGCG compared to the control group (no EGCG) and was concentration dependent.
(6) Influence of EGCG on Gene expression levels
qPCR detection: total RNA was extracted from EGCG treated Aeromonas hydrophila cultures using TransZol Up (TransGen Biotech, beijing, china) and cDNA synthesis was performed using reverse transcription kit (ThermoFisher Scientific, waltham, mass., USA) according to the manufacturer's instructions. The concentration and purity of the RNA were determined using an ultraviolet spectrophotometer (Implen, munich, germany). qRT-PCR in
480II Master (Roche, germany) was used and the final reaction was 10. Mu.L. The expression of the target gene was normalized to the expression of rpob, which was the housekeeping gene. The primer pairs used in this study are shown in Table 1 (hydroids) and Table 2 (zebra fish).
TABLE 1
TABLE 2
Experimental results: the effect of EGCG on Aeromonas hydrophila virulence factor and QS gene expression as shown in FIG. 6. All data in the figures represent three independent experiments, repeated three times each, expressed as mean ± SD of each histogram. * p <0.05; * P <0.01; * P <0.001.EGCG significantly inhibited the expression of ai-1 related genes ahyI and ahyR, but did not affect the expression of LuxR. LuxS is an AI-2 synthetase, and participates in the generation of AI-2 signal molecules, and EGCG significantly inhibits the expression thereof. EGCG significantly down-regulates the expression of virulence related genes including the hemolysin genes hly and ahh1, the flagella gene flgL and the elastase gene ahyB. This suggests that EGCG may inhibit expression of virulence genes by inhibiting the QS system.
Example 2 Effect of EGCG on protection of Aeromonas hydrophila on Zebra fish after infection with Aeromonas hydrophila
Animal maintenance: the clinic healthy wild adult zebra fish is collected from Chongqing fish farming house. All zebra fish were acclimatized in a sterile glass aquarium at 28±2 ℃ for 5 days and fed 2 commercial food particles per day. During the study, charcoal filtered fresh water was used.
(1) Protective effect of EGCG on aeromonas hydrophila infected zebra fish
Protection rate of EGCG against aeromonas hydrophila infection of zebra fish: aeromonas hydrophila was incubated to exponential phase in nutrient solution, washed 3 times with sterile PBS buffer, resuspended to 5X 108CFU/ml in PBS. Zebra fish were randomly divided into 5 groups (n=18) of vector, bacteria only and bacteria+egcg (10 mg/kg, 50mg/kg and 100 mg/kg). Bacteria (10. Mu.l, 5X 108 CFU/ml) were injected into the zebra fish peritoneal cavity and EGCG was injected. After bacterial challenge, zebra fish survival was recorded every 6 hours.
Experimental results: protection of the zebra fish against aeromonas hydrophila infection by EGCG as shown in fig. 7: (A) EGCG can alleviate symptoms of infection of zebra fish with aeromonas hydrophila, and (B) EGCG increases survival rate of zebra fish infected with aeromonas hydrophila. The survival rate of the uninfected control group is 100% after 48 hours, and the zebra fish starts to die after the aeromonas hydrophila is injected into the abdominal cavity for 6 hours. Dead zebra fish developed symptoms such as abdominal hemorrhage, ulcers and edema, and partial zebra fish developed symptoms such as ocular hemorrhage (fig. 7A). The death time of the first zebra fish injected with 50mg/kg and 100mg/kg EGCG was significantly delayed. Zebra fish injected with 50mg/kg EGCG for the first time died at 18h, and zebra fish injected with 100mg/kg EGCG for the first time died at 48h. The survival rate of the zebra fish in the model group is 0 after 48 hours of infection, and the survival rate of the zebra fish in the high and medium dose groups is as high as 88.9 percent. In contrast, the survival rate was the same for the low dose group as for the model group (fig. 7B).
(2) Adhesion of aeromonas hydrophila to zebra fish
Bacterial load: to quantify bacterial load, individual fish of each treatment group were homogenized and the slurry was made into a suspension. Mu.l of the serial dilution of zebra fish paste was applied to an RS medium (Haibo Bio Qingdao, china) plate, incubated three times at 30℃for 24h, colonies with a pronounced yellow color were counted, and the average bacterial load (CFU/ml) of Aeromonas hydrophila was calculated.
The effect of EGCG on the treatment group was assessed from the adherence of aeromonas hydrophila in the detoxified zebra fish. As shown in fig. 8, bacterial load of zebra fish was significantly reduced after EGCG treatment. All data represent three independent experiments, three replicates each, expressed as mean ± SD of each histogram. * p <0.05; * P <0.01; * P <0.001. The results show that the adhesion of aeromonas hydrophila to zebra fish is significantly reduced after EGCG treatment. The bacterial adhesion CFU was reduced by 21.22%, 33.47% and 38.00% in the EGCG groups at 10mg/kg, 50mg/kg and 100mg/kg, respectively, compared to the model group. Low concentrations of EGCG, while not improving the survival rate of zebra fish, still reduced bacterial adhesion.
(3) EGCG for inhibiting intestinal inflammation of zebra fish
Expression of zebra fish intestinal pro-inflammatory cytokine genes: the death rate of the zebra fish is sharply increased after the zebra fish is infected with aeromonas hydrophila for 6 hours, so the sampling time point is 6 hours. Intestinal tissues of zebra fish were collected rapidly. Gene expression of IL-1. Beta., IL-8, IL-6, TNF-alpha was detected by 1.8 qRT-PCR.
As shown in fig. 9, EGCG significantly reduced the production of zebra fish intestinal cytokines. All data represent three independent experiments, three replicates each, expressed as mean ± SD of each histogram. * p <0.05; * P <0.01; * P <0.001. After the aeromonas hydrophila is injected into the abdominal cavity for 6 hours, the intestinal tissues of the zebra fish show cytokine surge. However, EGCG treatment significantly inhibited the expression of the pro-inflammatory cytokine genes IL-1 beta, IL-6, IL-8 and TNF-alpha (FIG. 9), and the results indicate that EGCG can inhibit the strong inflammatory response caused by bacterial infection.
(4) Influence of EGCG on histopathology of post-challenge zebra fish
Histopathological analysis: for histopathological studies, zebra fish tissue specimens, such as gills, muscles, livers, were obtained from uninfected, infected and EGCG treated groups. Histological sections (5 μm) were prepared and stained with hematoxylin and eosin.
Histopathological examination showed that aeromonas hydrophila infection resulted in significant pathological changes. As shown in fig. 10, different groups of tissues were selected for histopathological analysis. Sections were stained with H & E. Inflammatory cell infiltration (green arrow), hepatocyte necrosis (black arrow), myofiber rupture (yellow arrow), gill silk lamina epithelial cell necrosis (red arrow).
In the model group, a large number of inflammatory cells infiltrated each tissue, indicating that aeromonas hydrophila infection resulted in a systemic inflammatory response. EGCG has a significant protective effect on the tissue of hydrophilic a.s arophila-infected zebra fish in a dose-dependent manner.
From the above experimental study, EGCG has a MIC of 1024. Mu.g/ml for Aeromonas hydrophila. The submicron of EGCG delays the time of aeromonas hydrophila entering the logarithmic growth phase, but has no obvious effect on the growth after 24 hours. Biofilms enable bacteria to withstand harsh environmental conditions such as starvation and desiccation, and cause a wide range of chronic diseases. Thus, biofilms are considered to be a major cause of persistent nosocomial infections in immunocompromised patients. The swimming activity of bacteria in a liquid environment depends on rotating polar and transverse flagella, which movement enables the bacteria to leave the hostile environment. EGCG (64-256. Mu.g/ml) does not directly produce bactericidal effects but inhibits biofilm formation, motility and colonisation and is concentration dependent compared to control (no EGCG). Haemolytic toxins are important causative factors of aeromonas infection. It forms a channel on the cell membrane of the target cell, resulting in cell death. In the studies of the present application, it was found that high concentrations of EGCG significantly inhibited the haemolytic activity of aeromonas hydrophila. The results show that EGCG at different concentrations significantly inhibited biofilm formation, viability and hemolysin production, suggesting that EGCG may interfere with virulence release by blocking the aeromonas hydrophila QS system. In addition, differential expression of virulence related genes was quantified using qRT-PCR. Among them, ahh and hly are key genes regulating the extracellular hemolytic capacity of Aeromonas hydrophila. The results show that ahh and hly gene expression is obviously reduced after EGCG treatment, and the EGCG treatment can be proved to weaken main virulence of aeromonas hydrophila. Furthermore, the present application also found down-regulation of flgL and ahyB gene expression following EGCG treatment. flgL is a gene encoding a flagellin, which is necessary for the adhesion and invasion of cells by hydrophilic bacteria, and a temperature stable metalloprotease ahyB with elastolytic activity plays an important role in the invasion and establishment of infections.
In order to search for EGCG specific interference pathway based on QS, the present application examined the relative expression levels of the ai-1 related gene ahyI, ahyR, luxR, ai-2 related gene LuxS and the ai-3 related gene QseB. Notably, EGCG significantly inhibited the expression of ahyI, ahyR, luxS and QseB. ahl mediated QS is believed to be an important factor in the virulence of some bacterial pathogens, promoting biofilm maturation, regulating extracellular enzyme and hemolysin production. AhyI and AhyR are homologs of the LuxI and LuxR quorum sensing proteins, and AhyRI systematically controls the expression of regulatory proteins associated with exonuclease activity. Both ahyI and ahyR mutations result in loss of serine and metalloprotease activity. In the studies of this application, EGCG significantly inhibited the expression of ahyI and ahyR, but had little effect on the homologous protein LuxR receptor. AI-3 is an amination product of aromatic compounds, an unusual mutual domain QS system, mainly found and elucidated in enterobacteria enterohemorrhagic escherichia coli. QseB is significantly inhibited by EGCG, and studies indicate that QseB is associated with biofilm, protease and hemolytic activity. Thus, the results of the EGCG gene expression analysis of the examples of the present application verify the results of physiological analysis, elucidating the potential of EGCG against QS.
In vitro experiment results show that EGCG can inhibit the generation of biomembrane and virulence factors mediated by vibrio hydrophila QS. To further demonstrate its effect, the examples herein performed in vivo experiments using the zebra fish aeromonas hydrophila infection model. Zebra fish are infected with aeromonas hydrophila by intraperitoneal injection. In vivo experimental results show that compared with a model group, EGCG remarkably improves the survival rate of zebra fish and reduces the infection rate of aeromonas hydrophila. Furthermore, EGCG treatment reduced zebra fish CFU count levels compared to untreated control groups. These results are consistent with Zhao Ling et al studying the protective effect of umbrella inflorescences on aeromonas hydrophila infected grass carp. Proinflammatory cytokines are commonly used to assess inflammation levels in vivo. After intraperitoneal injection, the zebra fish has ulcers and bleeding on the abdomen, and the number of pro-inflammatory cytokines in the model group is increased, which indicates that the vibrio hydrophila obviously induces the inflammation of the zebra fish. qRT-PCR results show that EGCG inhibits transcription of pro-inflammatory factor genes TNF-alpha, IL-1 beta, IL-8 and IL-6 in the intestinal tract. This means that EGCG may have the ability to inhibit excessive production of pro-inflammatory cytokines and excessive activation of the zebra fish innate immune system, thereby significantly reducing the mortality of severe bacterial infections. Consistent with this result, histopathological sections showed that EGCG significantly reduced inflammatory cell infiltration in the liver, muscle and gill of zebra fish. In the liver, the hepatic cord is disturbed, cells have different degrees of vacuolation or necrosis, muscle fibers break and dissolve, the gill tissue structure is destroyed, and epithelial cells on both sides of the gill wires are necrotized. EGCG can obviously improve the pathological damage caused by the hydrophila.
In conclusion, the submicron of the EGCG can remarkably inhibit the toxicity of aeromonas hydrophila, improve the survival rate of the zebra fish infected with aeromonas hydrophila, effectively reduce the bacterial load and reduce the inflammatory reaction. The QS system may be a potential pathway for EGCG to reduce aeromonas hydrophila virulence. EGCG is therefore a promising QSI for alleviating aeromonas hydrophila infection.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (8)
1. Application of epigallocatechin-3-gallate in preparing quorum sensing inhibitor of aeromonas hydrophila is provided.
2. The application of epigallocatechin-3-gallate in preparing active preparation for inhibiting aeromonas hydrophila biomembrane, hemolysin, swimming and surging virulence factors.
3. The application of epigallocatechin-3-gallate in inhibiting the quorum sensing related gene expression and virulence factor related gene expression of aeromonas hydrophila.
4. Use of epigallocatechin-3-gallate for inhibiting quorum sensing-related gene expression and virulence factor related gene expression in aeromonas hydrophila as defined in claim 3, wherein said quorum sensing-related genes include ahyI, ahyR, luxS and QseB; the virulence factor related genes include hemolysin genes hly and ahh1, flagella gene flgL and elastase gene ahyB.
5. The application of epigallocatechin-3-gallate in preparing medicine for treating or preventing organism infection of Aeromonas hydrophila is provided.
6. The use of epigallocatechin-3-gallate in the manufacture of a medicament for treating or preventing infection of an organism with aeromonas hydrophila as claimed in claim 5 wherein said organism is a zebra fish.
7. A quorum sensing inhibitor of aeromonas hydrophila, wherein the active ingredient of the quorum sensing inhibitor comprises epigallocatechin-3-gallate.
8. A medicament for treating or preventing infection of an organism with aeromonas hydrophila, wherein the active ingredient of the medicament comprises epigallocatechin-3-gallate.
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