Application of curcumin and quercetin combined medicine in preparation of medicine for treating fish nocardia disease

Abstract

The invention discloses application of curcumin and quercetin combined medicine in preparation of a medicine for treating fish nocardia disease, and belongs to the technical field of aquaculture. The invention develops a new application of curcumin and quercetin combined medicine in the field of aquaculture, and has a wide potential application prospect. On one hand, curcumin and quercetin can be directly used as drugs for preventing and treating nocardia; the curcumin and quercetin combined drug can also be used as a feed additive to prevent and treat nocardiasis infection of fishes. The curcumin and quercetin combined drug provided by the invention has the characteristics of high efficiency, safety, no residue, easiness in degradation, low drug resistance and the like, and provides a new treatment method and way for fish nocardia infection.

Description

TECHNICAL FIELD

[0001] The application relates to the field of aquaculture technology, and particularly relates to application of curcumin and quercetin combination medication in preparation of fish nocardiosis treatment drugs. BACKGROUND

[0002] Nocardiosis is a chronic infectious disease caused by Nocardia, which is a zoonosis, and poses a great threat to aquaculture and aquatic product safety. Nocardia is a gram-positive bacterium widely distributed in soil and water environments, and the Nocardia that invades fish mainly includes Nocardia seriolae, N. asteroides and N. salmonicida. Among them, Nocardia seriolae is the most common pathogenic bacterium of fish, which can cause a wide range of nodules in internal organs and body surface, body surface ulceration, hemorrhage and other symptoms, and has the characteristics of high infection rate and high mortality. Although antibiotics were once the main control means, due to the blockage of granulomas and the increase of antibiotic resistance, the prevention and control effect of the disease in actual production is poor. Therefore, it is of great importance to develop safe and efficient targeted therapeutic drugs for the sustainable development of aquaculture.

[0003] Chinese herbal medicine has the advantages of easy degradation and difficulty in developing drug resistance, and can be used in fish disease prevention and control. The prior art reports that Magnolia officinalis extract has a preventive and therapeutic effect on nocardiosis, but the application of curcumin and quercetin combination medication in the prevention and treatment of nocardiosis has not been disclosed. SUMMARY

[0004] In order to solve the above-mentioned deficiencies existing in the prior art, the purpose of the present application is to provide application of curcumin and quercetin combination medication in preparation of fish nocardiosis treatment drugs.

[0005] The technical scheme for solving the above-mentioned technical problems is as follows: application of curcumin and quercetin combination medication in preparation of fish nocardiosis treatment drugs or fish feed additives or as a targeted Nocardia ICL receptor inhibitor.

[0006] On the basis of the above technical scheme, the present application can also be improved as follows:

[0007] Further, the fish nocardiosis is caused by Nocardia seriolae, N. asteroides or N. salmonicida.

[0008] Further, the fish is a conventional aquaculture fish, such as Micropterus salmoides.

[0009] Further, the mass ratio of curcumin and quercetin in the curcumin and quercetin combination medication is 1-5:1-5; preferably, the mass ratio of curcumin and quercetin is 1:1.

[0010] Further, the dosage form of the Nocardia fish disease treatment drug is an oral preparation, a soaking agent or an injection.

[0011] Further, the Nocardia fish disease treatment drug further comprises a pharmaceutically acceptable adjuvant, and the adjuvant comprises at least one of a solubilizer, a stabilizer or a bait binder for aquatic products.

[0012] Further, the minimum inhibitory concentration of the Nocardia fish disease treatment drug to fish Nocardia is ≤4 μg / mL.

[0013] A Nocardia ICL receptor targeting inhibitor comprises curcumin and / or quercetin.

[0014] The present application has the following beneficial effects:

[0015] The present application has the following beneficial effects:

[0016] The present application opens up a new use of curcumin and quercetin combination therapy in the field of aquaculture, and has a broad potential application prospect. On the one hand, curcumin and quercetin can be directly used as drugs for preventing and treating Nocardia; and on the other hand, curcumin and quercetin combination therapy can be used as a feed additive to prevent and treat fish Nocardia infection.

[0017] The curcumin and quercetin combination therapy provided by the present application has the characteristics of high efficiency, safety, no residue, easy degradation and low drug resistance, and provides a new treatment method and approach for fish Nocardia infection. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The three-dimensional structure of ICL of Nocardia fish and its conformation rationality obtained by different modeling methods are evaluated; wherein, A is the SWISS-MODEL homology modeling of ICL of Nocardia fish; B is the Phyre2 homology modeling of ICL of Nocardia fish; C is the Robetta homology modeling of ICL of Nocardia fish; D is the SWISS-MODEL evaluation of the rationality of the main chain structure of ICL; E is the Phyre2 evaluation of the rationality of the main chain structure of ICL; and F is the Robetta evaluation of the rationality of the main chain structure of ICL.

[0019] Figure 2Figure 2 is a 2D interaction diagram of five in-vitro bacteriostatic small molecule compounds with ICL receptors; wherein, Figure A is a 2D structure interaction diagram of curcumin with ICL receptors; Figure B is a 2D structure interaction diagram of chrysophanol with ICL receptors; Figure C is a 2D structure interaction diagram of baicalein with ICL receptors; Figure D is a 2D structure interaction diagram of berberine with ICL receptors; Figure E is a 2D structure interaction diagram of quercetin with ICL receptors; wherein, the green dotted line represents a hydrogen bond, and the arc with a short radial line represents a hydrophobic interaction.

[0020] Figure 3 Figure 3 is an effect of curcumin and quercetin combination medication on the activity of Nocardia; wherein, Figure A is the chemical formula and three-dimensional structure of curcumin and quercetin; Figure B is the growth curve after curcumin and quercetin combination medication (0-8 μg / mL) treatment; Figure C and D are the changes in bacterial activity after 72 h treatment with different concentrations of curcumin and quercetin combination medication compared with the DMSO group (CCK-8, n=3) (P<0.01 is extremely significant, florfenicol (2 μg / mL) is a positive control group); Figure D is the gram staining morphology of untreated Nocardia; Figure E is the gram staining morphology of Nocardia after combination medication (MIC concentration) treatment; *P<0.05 represents a significant difference; ** or ***P<0.001 represents an extremely significant difference.

[0021] Figure 4 Figure 4 is the effect of curcumin and quercetin combination medication on the cell wall thickness of Nocardia.

[0022] Figure 5 Figure 5 is the effect of different concentrations of medication on the alkaline phosphatase activity in the supernatant of Nocardia.

[0023] Figure 6 Figure 6 is the effect of curcumin and quercetin combination medication on the biofilm of Nocardia; wherein, Figure A is the effect of different concentrations of curcumin and quercetin combination medication on the biofilm formation of Nocardia; Figure B is the inhibition rate of different concentrations of curcumin and quercetin combination medication on the biofilm formation of Nocardia compared with the DMSO group; Figure C is the ability of different concentrations of curcumin and quercetin combination medication to remove the biofilm of Nocardia; Figure D is the removal rate of different concentrations of curcumin and quercetin combination medication on the biofilm of Nocardia compared with the DMSO group; different superscript letters represent a significant difference.

[0024] Figure 7 Figure 7 is the effect of curcumin and quercetin on the activity of ICL.

[0025] Figure 8 Figure 8 is the clinical sign results of Micropterus salmoides in different treatment groups.

[0026] Figure 9Statistical analysis of mortality and internal nodule number of largemouth bass in different treatment groups; Wherein, A figure is the cumulative mortality of largemouth bass in different treatment groups within 14 days; B figure is the statistical analysis of the number of white nodules on the head kidney, spleen and liver of the infection group and the treatment group (14d, n=3).

[0027] Figure 10 Histopathological observation (H&E) and pathological score of the head kidney of largemouth bass in different treatment groups; Wherein, A figure is the blank control group; B figure is the DMSO solvent group; C figure is the nocardia infection group, extensive and large area of mature granuloma area (black dotted circle), C" shows the local enlargement of granuloma, a large number of macrophages, neutrophil infiltration, and severe cell necrosis (black arrow); D figure is the combined drug treatment group, showing focal and small area of early granuloma (white dotted circle), D" shows the local enlargement of granuloma, showing a small amount of similar cell infiltration, and the granuloma is not obvious; E figure is the comparison of the number of granulomas in the head kidney HE staining of the infection group and the treatment group (statistical area 1x1mm, n=3); F figure is the overall pathological score of the head kidney HE staining of the infection group and the treatment group.

[0028] Figure 11 Histopathological observation (H&E) and pathological score of the spleen of largemouth bass in different treatment groups; Wherein, A figure is the blank control group; B figure is the DMSO solvent group; C figure is the combined drug treatment group, a large number of melanin macrophages (white arrow), a small number of granulomas, C' shows the local enlargement of granuloma, showing inflammatory cell infiltration and granuloma in the development stage; D figure is the nocardia infection group, a large number of mature granulomas (black dotted line); E figure is the local enlargement of the spleen of the treatment group, showing splenic blood (white dotted square); F figure is the glass-like degeneration of blood vessels in the infection group (black arrow), with vascular dilation and similar cell infiltration; G figure is the local enlargement of granuloma in the infection group, with caseous necrosis in the central area (black arrow); H figure is the comparison of the number of granulomas in the spleen HE staining of the infection group and the treatment group and the overall lesion score of the spleen (n=3).

[0029] Figure 12Figure 6 shows the histopathological observation (H&E) and pathological score of the liver of different treatment groups of largemouth bass; wherein, Figure A is the blank control group; Figure B is the DMSO solvent group; Figure C is the treatment group, no obvious lesions are observed; Figure D is the nocardia infection group, D' shows the local enlargement of the granuloma of the infection group, and the central area shows obvious caseous necrosis (black-tailed arrow); Figure E is the local area enlargement of the treatment group, a small amount of class cell infiltration can be seen in the blood vessels (white-tailed arrow); Figure F is the local enlargement of the infection group, hepatocyte degeneration and necrosis, and blood vessels are congested (no-tailed black arrow); Figure G is the local enlargement of the infection group, a large number of neutrophils are infiltrated around the blood vessels (black dotted circle); Figure H is the comparison of the number of granulomas in the spleen HE staining of the infection group and the treatment group and the overall lesion score of the spleen (n=3).

[0030] Figure 13 Figure 5 shows the determination results of serum enzyme activities of different treatment groups; wherein, Figure A is the comparison of lysozyme activities of different treatment groups at different time points; Figure B is the comparison of acid phosphatase activities of different treatment groups at different time points; Figure C is the comparison of superoxide dismutase activities of different treatment groups at different time points; Figure D is the comparison of catalase activities of different treatment groups at different time points; Figure E is the color of serum of each group on the 5th day after challenge; Figures F-G are the colors of serum on the 8th day after challenge and treatment, respectively; *P<0.05 represents significant difference; ***P<0.001 represents extremely significant difference.

[0031] Figure 14 Figure 4 shows the evaluation results of the bacterial load of the head kidney tissue of different treatment groups; wherein, Figure A is the blank control group; Figure B is the DMSO solvent group; Figure C is the treatment group; Figure D is the infection group; Figure E is the comparison of the bacterial load of the head kidney tissue of the four groups (n=3).

[0032] Figure 15 Figure 3 shows the determination results of the inflammatory factors of the head kidney of different treatment groups; wherein, Figures A-B are the expression differences of the pro-inflammatory factors of different treatment groups; Figures C-D are the expression differences of the anti-inflammatory factors of different treatment groups; Figures E-F represent the head kidney of the blank control and the DMSO group, respectively; Figure G is the head kidney of the challenge group; Figure H is the head kidney of the treatment group; *P<0.05 represents significant difference; **P<0.01 represents extremely significant difference. DETAILED DESCRIPTION

[0033] The glyoxylate cycle is a key metabolic pathway for bacteria to survive under nutritional deficiency and environmental stress, in which isocitrate lyase (ICL) as the core enzyme of the cycle can utilize fatty acids as carbon source to maintain bacterial function and survival. The present application takes ICL of Nocardia seriolae as the target, and obtains candidate small molecules by using structure-based virtual screening technology, and evaluates the pharmacological effect of the obtained compounds by in vitro antibacterial activity detection, biofilm experiment, in vivo protection effect, etc. to obtain small molecule compounds for targeted inhibition of Nocardia seriolae disease. The specific experiment is as follows:

[0034] Example 1: Construction of three-dimensional model of ICL receptor of Nocardia seriolae

[0035] The ICL receptor of Nocardia seriolae is constructed by homology modeling technology, specifically:

[0036] (1) Use online resources such as SWISS-MODEL, Phyre2 and Robetta to generate multiple homology models, and screen the optimal three-dimensional conformation by the Ψ(psi) dihedral angle of the amino acid residues in the peptide bond;

[0037] (2) Further score the optimal model by using the ERRAT Complete evaluation standard in the SAVES network server;

[0038] (3) After determining the docking model, use PyMOL software to pretreat the three-dimensional model of the receptor (residue protonation, addition of hydrogen atoms, removal of water molecules, merging of nonpolar hydrogen, deletion of lone pair electrons, and removal of non-standard amino acid chains) to obtain an optimized object suitable for virtual screening;

[0039] (4) Use MGLtools to import the PDB format file into MGLtools and convert it into a PDBQT format file.

[0040] The present application constructs a three-dimensional structural model of the ICL receptor by online tools such as SWISS-MODEL, Phyre2 and Robetta. Although different prediction algorithms are used by each tool, the generated ICL receptor structures all show high consistency. To further verify the quality of the model, all predicted models are analyzed using the SAVES network server. The Ramachandran Plot evaluation results show that the main chain structures of the three models are highly reasonable. In the SWISS-MODEL model, 92.8% of the amino acid residues are located in the most favorable region (red region), 6.9% are located in the allowed region (yellow region), and no residues are located in the disallowed region (white region); in the Phyre2 model, 92.3% of the amino acid residues are located in the most favorable region, 7.4% are located in the allowed region, and no residues are located in the disallowed region; in the Robetta model, 92.1% of the amino acid residues are located in the most favorable region, 7.7% are located in the allowed region, and no residues are located in the disallowed region (see Figure 1 ) for details.

[0041] To further evaluate the quality of the three-dimensional model constructed by SWISS-MODEL, the present application uses the ERRAT program to analyze the non-bonding interactions between different atom types in the model. The comprehensive score of the model is 95.962, indicating that it performs well in non-bonding interactions. That is, the ICL receptor model generated by SWISS-MODEL not only has more reasonable main chain structure, but also has excellent non-bonding interaction performance. Therefore, the protein model constructed by SWISS-MODEL is used as the ICL receptor model in the present application.

[0042] Example 2: In vitro antibacterial experiment of candidate small molecules

[0043] Using the three-dimensional model of the ICL receptor of N. hereina constructed by SWISS-MODEL, 27 small molecules with binding energy less than -9 kcal / mol and good drug-like properties are obtained by virtual screening, and in vitro antibacterial experiments are performed, as follows:

[0044] I. Experimental strain

[0045] Isolated from large-mouth bass with sarcoidosis, further identified as N. hereina by physiological and biochemical tests and 16S rRNA sequencing.

[0046] II. 27 experimental drugs

[0047] Curcumin, oleanolic acid, naringenin, corydalis tuber base, ellagic acid, lovastatin, phloretin, chrysophanol, chelidonine, andrographolide, phlorizin, imatinib, apigenin, rotenone, linagliptin, baicalein, sinomenine, cucurbitacin B, chlorogenic acid, ursolic acid, berberine, limonin, evodiamine, chelidonine, myricetin, quercetin, seneginic acid.

[0048] III. Experimental Methods

[0049] The MIC determination method was used to conduct in vitro antibacterial experiments on 27 kinds of small molecule compounds, and the specific steps were as follows:

[0050] (1) Prepare 96-well plates: In the 2nd-12th columns of the 96-well plate, add 100 μL of BHI liquid medium to each well as a dilution solution.

[0051] (2) Prepare drug solution: Dilute each small molecule stock solution (10 μM) dissolved in DMSO with BHI liquid medium, then add the diluted stock solution to the first column of the 96-well plate, so that the final total volume of each well is 200 μL.

[0052] (3) Two-fold dilution method: Starting from the first column of the 96-well plate, use the two-fold dilution method to sequentially dilute back to the 12th column to form a series of small molecule solutions with different concentrations.

[0053] (4) Set up control groups: The negative control does not need to add bacteria, only contains small molecule drugs and medium; the positive control does not add drugs, only contains Nocardia cruenta liquid and medium.

[0054] (5) Inoculate bacterial suspension: Dilute the bacterial suspension to 3 x 10 7 CFU / mL, then add 100 μL of the bacterial suspension to each well of the 1st-12th columns of the 96-well plate, so that the final concentration of small molecules in the first column of each well is 128 μg / mL.

[0055] (6) Incubation conditions: Place the 96-well plate in a 28°C incubator and incubate for 24 h.

[0056] (7) Detection endpoint: After incubation, add 20 μL of resazurin aqueous solution to each well, and incubate at 28°C for 4 h. Observe the color change in the well, and take the lowest concentration with no change to pink as the MIC value of the small molecule.

[0057] From the test results, it can be seen that Nocardia iowensis shows strong resistance to most compounds, including more than 20 small molecule compounds such as oleanolic acid, naringenin, corydalis peashrub alkaloid, and gallic acid. In contrast, the five small molecule compounds of curcumin, chrysophanol, baicalein, berberine and quercetin all show significant antibacterial activity. Among them, the minimum inhibitory concentration (MIC) of curcumin, chrysophanol, baicalein and quercetin is 32 μg / mL, and the MIC of berberine is 64 μg / mL, and the MIC of the remaining compounds is greater than 128 μg / mL.

[0058] In order to verify whether the five small molecule compounds obtained by the above screening can stably bind to the ICL target and thus play an antibacterial role, the LIGPLOT+ program (parameter settings: ) was used to analyze the interaction between the five small molecule compounds and the ICL receptor, and a two-dimensional interaction diagram was generated, which is specifically shown in Figure 2 .

[0059] As can be seen from Figure 2 , curcumin and quercetin have more strong interactions (hydrogen bonds) with the ICL target, indicating that these two compounds have strong binding capacity with the ICL receptor. In contrast, the other three compounds have less interaction with the ICL receptor, indicating that their binding stability is relatively low.

[0060] Therefore, curcumin and quercetin were combined, and their synergistic antibacterial effect was verified according to the above in vitro antibacterial experiment process. Specifically, curcumin and quercetin were combined at a mass concentration ratio of 1:1, and the broad-spectrum antibiotic florfenicol was used as a positive control to evaluate the synergistic antibacterial effect. From the antibacterial results, it can be seen that the MIC of curcumin and quercetin combined for Nocardia iowensis is 4 μg / mL, showing obvious synergistic antibacterial effect, which is significantly lower than the minimum inhibitory concentration of 32 μg / mL when used alone.

[0061] Example 3: In vitro antibacterial experiment of curcumin and quercetin combined with drugs

[0062] This example describes the in vitro antibacterial activity of curcumin and quercetin combined with drugs on Nocardia iowensis, as follows:

[0063] I. Bacterial activity determination

[0064] Nocardia iowensis was diluted to 3 x 10 7CFU / mL, 200 μL of the prepared bacterial suspension was added to a 96-well plate, and different concentrations of curcumin and quercetin were added to achieve final concentrations of 1 / 2 MIC (2 μg / mL), MIC (4 μg / mL), and 2 MIC (8 μg / mL) respectively (small molecules were dissolved by sonication using DMSO). The following control groups were set up: blank control group (containing only BHI medium), solvent control group (containing only DMSO), and positive control group (florfenicol, 2 μg / mL), with three replicates for each group.

[0065] The 96-well plates were incubated statically at a constant temperature of 28℃, and the absorbance at 600 nm was measured using a microbial growth curve analyzer at time points of 0, 4, 8, 12, 24, 48, 72, 96, and 120 h. Growth curves were then plotted with time on the x-axis and absorbance on the y-axis to evaluate the effect of different treatment concentrations on the growth of Nocardia auricula-judae.

[0066] To further investigate the effect of combined drug therapy on Nocardia purpurea cell viability, bacteria were inoculated into BHI medium and cultured at 28°C with shaking at 180 rpm for 24 h. Then, 200 μL of the bacterial culture was transferred to a new blank 96-well plate to achieve final drug concentrations of 1 / 2 MIC, MIC, and 2 MIC. A DMSO solvent control group was also included. Subsequently, 20 μL of CCK-8 reagent was added to each well, and the plates were incubated at 28°C for 24 h. Finally, the absorbance of each well was measured at 495 nm to quantitatively analyze cell viability. The cell viability calculation formula is as follows: (Ab... 实验组 -Ab 空白组 ) / (Ab 对照组 -Ab 空白组 )×100%, where Ab represents the absorbance value.

[0067] The test results showed that when the concentration reached the MIC and above, the combined use of drugs significantly inhibited the growth of Nocardia auriculata. Figure 3 (See Figure B in the table). It also showed some antibacterial effect at a lower concentration of 1 / 2 MIC. As the drug concentration increased to 2 MIC, the bacterial growth inhibition showed a clear concentration-dependent effect, and its antibacterial effect was comparable to the positive control florfenicol (2 μg / mL). Further analysis using a CCK-8 assay kit to detect the cell viability of Nocardia after treatment with different concentrations (2-8 μg / mL) of the combined drug revealed that, compared with the DMSO negative control group, the combined drug treatment significantly reduced bacterial viability, showing a concentration-dependent decreasing trend (P<0.01). Figure 3 (Figure C in the diagram); among them, the bacterial activity in the 2MIC treatment group was the lowest, similar to that in the florfenicol treatment group. Furthermore, Gram staining results showed significant morphological changes in the bacteria after drug treatment, manifested as lighter staining and reduced branching. Figure 3D, E in FIG. 1.

[0068] II. Transmission electron microscopy observation

[0069] The sample was prepared by ultrathin sectioning technique and observed under transmission electron microscope to investigate the effect of combined drug use on the ultrastructure of Nocardia seriolae. The transmission electron microscopy observation results show that the Nocardia seriolae in the DMSO negative control group presents a typical dendritic structure, the edge is irregular, the boundary between the cell wall and the cell membrane is clear, and the thickness is uniform. The dense area can be seen in the cytoplasm, which is the nucleoid area where the double-stranded DNA is located, and the loose transparent area around the nucleoid is the cytoplasmic area containing rich ribosomes and metabolic products or storage particles. In contrast, after CQ (MIC) treatment, the bacterial morphology becomes blunt, the boundary between the cell wall and the cell membrane is blurred and uneven, and in some areas, the cell wall seems to have cracks or damage, in addition, the distribution of substances in the cytoplasm is uneven, some areas appear relatively dense, and other areas are relatively sparse, and some granular structures can be observed, which are probably damaged organelles or metabolic products.

[0070] III. Cell wall integrity assay

[0071] In bacteria, alkaline phosphatase is usually located in the periplasmic space, which is a region between the bacterial cell wall and the cell membrane, so the present application evaluates the effect of combined drug use on the integrity of bacterial cell wall and membrane by detecting the alkaline phosphatase activity in the supernatant. The specific operation steps are as follows:

[0072] (1) Prepare bacterial solution: collect bacterial cells from the logarithmic growth phase culture of Nocardia seriolae, wash with sterile PBS buffer (0.01 mol / L, pH 7.2), and resuspend three times by centrifugation (5000 rpm, 10 min) to remove metabolic products and other substances that may interfere with the experimental results in the old culture medium, and finally adjust the concentration of bacterial cells to 3×10 7 CFU / mL.

[0073] (2) Drug treatment: divide the prepared bacterial solution into 15 mL centrifuge tubes, and add combined drugs with final concentrations of 1 / 2 MIC, MIC and 2 MIC as treatment groups, and set up DMSO control group by adding equal amount of DMSO.

[0074] (3) Incubation: incubate the samples containing different treatment conditions at 28°C for 4h to allow the drugs to interact with the bacteria.

[0075] (4) Collect the supernatant: after the incubation is completed, collect the supernatant of each group by centrifugation (5000 rpm, 10 min) again, which will be used for subsequent alkaline phosphatase activity assay.

[0076] (5) Alkaline phosphatase activity assay: The AKP assay kit was used to measure the alkaline phosphatase activity of the supernatant in each sample according to the manufacturer's instructions.

[0077] (6) Data analysis: To ensure the reliability and repeatability of the data, the entire experiment should be repeated at least three times.

[0078] To further evaluate the effect of curcumin combined with quercetin on the cell wall of Nocardia seriolae, the cell wall thickness was measured using Image J software, and the results showed that the cell wall thickness was significantly reduced after drug treatment (P < 0.001) Figure 4 ). At the same time, the alkaline phosphatase activity of the bacterial supernatant was significantly increased (P < 0.001), indicating that the combined use of drugs caused significant damage to the bacterial cell wall Figure 5 ).

[0079] Four, determination of biofilm inhibition and elimination effect

[0080] 1. Biofilm inhibition experiment

[0081] After diluting the overnight culture of Nocardia seriolae to 3 x 10 7 CFU / mL using BHI liquid medium, 200 μL was inoculated into a 96-well plate. Three groups of controls were set up: a blank control group (containing only the culture medium), a DMSO solvent control, and a positive control of 2 μg / mL florfenicol. All samples were incubated at 28°C, and to evaluate the effect of curcumin combined with quercetin, mixed solutions of 1 / 2 MIC, MIC, and 2 MIC concentrations were prepared and added to the corresponding wells. After 24 h, the biofilm formation in each well was observed and evaluated, and the specific steps were as follows: after removing the culture solution, the plate was gently washed three times with PBS and allowed to air dry. Subsequently, 200 μL of glutaraldehyde (2.5% concentration) was added to each well and fixed for 90 min, and then washed twice. Then, 200 μL of crystal violet solution (0.1% concentration) was added to each well, and after staining for 15 min, it was washed twice with PBS and allowed to air dry. Finally, 200 μL of 95% ethanol was added to each well to dissolve for 30 min, and the absorbance value was measured at a wavelength of 550 nm using a UV spectrophotometer to quantify the amount of biofilm.

[0082] 2. Biofilm removal experiment

[0083] The Nocardia seriolae in the logarithmic growth phase was diluted to 3 x 10 7CFU / mL and inoculated 200 μΐ into 96-well plates, with blank control group (containing only BHI liquid medium) and DMSO solvent group. Incubated at 28°C for 24 h, after incubation, the bacterial suspension was carefully removed and washed twice with PBS to remove unattached bacteria. Subsequently, 1 / 2 MIC, MIC and 2 MIC concentrations of curcumin and quercetin combination solution were added to each well, and the positive control group was added with 2 μg / mL florfenicol. All samples continued to be incubated under the same conditions for 24 h. After incubation, the supernatant was discarded, and washed again with PBS and air dried. 200 μΐ of 2.5% glutaraldehyde was added to each well for 90 min, and then the fixing solution was discarded and washed with PBS several times. Then, 200 μΐ of crystal violet (0.1% concentration) was added to each well and stained for 15 min, and then the dye was discarded and washed twice with PBS. Finally, 200 μΐ of 95% ethanol was added to each well for 30 min, and the absorbance value was measured at 550 nm wavelength using a UV spectrophotometer to quantify the amount of biofilm. Biofilm inhibition / clearance rate = 1- (Abtest / Ab control ) x 100%, where Abtest represents the absorbance value of the test sample, and Ab control is the absorbance value of the control group.

[0084] Biofilm inhibition experiments showed that at 1 / 2 MIC (2 μg / mL), MIC (4 μg / mL) and 2 MIC (8 μg / mL) concentrations, the combination of curcumin and quercetin could inhibit the formation of Nocardia biofilm in a concentration-dependent manner Figure 6 (A figure in the middle). In particular, at MIC and above concentrations, the effect of this combination was comparable to the florfenicol group, both showing strong biofilm inhibition ability, and the crystal violet staining image further proved this conclusion Figure 6 (B figure in the middle).

[0085] Biofilm clearance experiments showed that the florfenicol group did not show obvious biofilm clearance effect, but the combination of curcumin and quercetin showed significant biofilm clearance ability at MIC and above concentrations, and this clearance effect also showed concentration dependence Figure 6 (C, D figures in the middle). This shows that the combination of curcumin and quercetin can not only effectively inhibit the formation of biofilm in vitro, but also significantly clear the formed biofilm.

[0086] V. ICL activity detection

[0087] ICL catalyzes the conversion of isocitrate to glyoxylate. Glyoxylate and NADH (the reduced form of nicotinamide adenine dinucleotide) react with LDH (lactate dehydrogenase) to form NAD (nicotinamide adenine dinucleotide). NADH has a characteristic absorption peak at 340 nm, and changes in absorbance at 340 nm can indirectly reflect ICL activity. Therefore, this invention evaluated the changes in ICL activity in Nocardia auricula-judae after 4 hours of combined drug treatment. The experiment was divided into a control group, a single-drug group, and groups treated with different concentrations of combined drugs. The specific operating steps are as follows:

[0088] (1) Bacterial treatment: After centrifugation, discard the supernatant and collect the bacterial cells into centrifuge tubes. According to the requirement of processing 5 million bacteria per 1 mL of extract, the bacteria were disrupted by ultrasonication (set power 200W, each ultrasonication time is 3s, the interval is 10s, and it is repeated 30 times). After centrifugation at 15000 rpm and 4℃ for 20 min, the supernatant was collected and placed on ice for testing.

[0089] (2) Before using the UV spectrophotometer, turn it on and warm it up for more than 30 minutes, set the absorption wavelength to 340nm, and calibrate the instrument with distilled water.

[0090] (3) According to the instructions of the ICL activity assay kit (AC10377, Shanghai Jizhi), add the reagents to a 1mL quartz cuvette in sequence. Start timing at the same time as adding reagent 6. Record the initial absorbance A1 at 10 seconds at a wavelength of 340nm. After colorimetric measurement, quickly place the cuvette and the reaction solution into a 25℃ water bath for accurate reaction for 2 minutes. Quickly remove the cuvette and wipe it dry. Measure the absorbance A2 at 2 minutes and 10 seconds at 340nm. Calculate ΔA = A1 - A2.

[0091] (4) Definition of enzyme activity unit: One enzyme activity unit is expressed as 1 nmol of NADH consumed per minute by 10,000 bacteria in the reaction system, i.e.: ICL (U / 10 4 cell)=ΔA÷(ε×d)×V_total×10 9 ÷(500÷Vsample×Vsample)÷T=4.59×ΔA.

[0092] The above states that the total reaction volume (Vreaction) is 1.0 × 102. -3 L; V: The volume of sample added is 0.035 mL; ε: The molar extinction coefficient of NADH is 6.22 × 10⁻⁶. 3 L / mol / cm; d: optical path length of the quartz cuvette is 1 cm; V_extract: volume of extract added is 1 mL; 500: bacterial count is 5 million; T: reaction time is 2 min; 10 9 Unit conversion factor, 1mo1 = 10 9 nmo1.

[0093] From the results, it can be seen that curcumin and quercetin (both at a concentration of 32 μg / mL) can significantly inhibit the activity of ICL; among them, quercetin has a more prominent inhibitory effect on ICL activity. When the total concentration of the combination drug is more than 4 μg / mL, the inhibitory effect on ICL activity shows a certain concentration-dependent characteristic. The above findings show that curcumin and quercetin can inhibit the activity of ICL, and have a synergistic effect on the inhibition of ICL when used in combination (when the concentration is above 4 μg / mL) Figure 7 , and the superscripts represent significant differences between the two groups.

[0094] Example 4: In vivo animal experiment of combination drug

[0095] This example describes the protective effect of curcumin and quercetin combination drug on Nocardia seriolae infection in large-mouth bass, as follows:

[0096] I. LC 50 determination

[0097] Prepare 7 transparent small glass jars, each containing 2 L of tap water and fully aerated for 24 h for standby.

[0098] Add curcumin and quercetin stock solution dissolved in DMSO to each jar (concentration of 10 μM) so that the final concentration of curcumin and quercetin in each jar is 128 mg / L, 64 mg / L, 32 mg / L, 16 mg / L, 8 mg / L, 4 mg / L and 0 mg / L. Then put 10 large-mouth bass of the same size into each jar, and set the experimental period to 96 h, during which no feed is provided and continuous aeration is maintained. Record the daily mortality rate and observed clinical symptoms and gross pathological changes, and calculate the 96 h LC 50 of curcumin and quercetin combination drug on large-mouth bass using the probit method.

[0099] LC 50 The results show that the mortality rate of large-mouth bass shows a significant concentration-dependent relationship with the combination drug (P < 0.001). Specifically, the DMSO control group and Group = 4 (4 μg / mL concentration) did not show any mortality during the entire observation period; however, in Group = 128, all large-mouth bass died within 12 h; the 96 h cumulative mortality rate in Group = 8 was only 33%. The probit method was used to calculate that the 96 h LC 50 of curcumin and quercetin combination drug on large-mouth bass was 24 mg / L.

[0100] II. Injection of challenge and treatment

[0101] From 400 large mouth bass randomly selected 360 tail without injury, healthy and lively, the average weight of 15.3±0.15g of large mouth bass, average distribution to 12 glass jars of potassium permanganate disinfection. Randomly divided into four groups: infection group, treatment group, solvent control group and blank control group, each group contains three repeated samples, each sample contains 30 fish. All glass jar water volume is 480L, and the oxygen supply is ensured by using aerator. The experimental group and treatment group use intraperitoneal injection method to attack bacteria, and the attack concentration is adjusted to 3×10 7 CFU / mL. Within four days after infection, white nodules began to appear in the viscera, and the treatment group was fed with small molecule combined drugs with body weight (3%) MIC concentration; The solvent control group only added the same amount of DMSO during the whole experiment; While the blank control group did not add any substance throughout the process. The water temperature was maintained at about 28℃ during the whole experiment, and no feed was given. The whole experimental period was set as 14d; The mortality of each group was recorded daily, and the dead individuals were removed in time.

[0102] The results show that during the 14-day observation period, the large mouth bass in the four different treatment groups (Control represents blank control, DMSO represents solvent control; N.S represents the infection group; N.S+CQ represents the treatment group with drug concentration of 4μg / mL) show significant differences in clinical symptoms. The appearance of large mouth bass in the blank control group and DMSO control group is normal, and no lesions are found; In contrast, the large mouth bass in the infection group showed symptoms such as anal redness and bleeding, dorsal fin granuloma and tail fin rot; While the surface of the large mouth bass in the treatment group showed no obvious abnormalities( Figure 8 ). Further anatomical examination found that there were a large number of white nodules in the head kidney, spleen and liver of the infection group of large mouth bass, indicating that the infection was severe; In contrast, the number of white nodules in the treatment group was significantly reduced, indicating that the combined therapy of curcumin and quercetin had a positive effect on inhibiting disease progression.

[0103] Survival curve analysis shows that during the entire 14-day experimental period, no large mouth bass was found in the blank control group and the DMSO solvent group; In contrast, the infection group began to die from the 4th day, and the cumulative mortality rate was close to 90% by the end of the experiment; While the treatment group also began to record deaths from the 4th day, but its 14-day cumulative mortality rate was only about 30%, which was significantly lower than that of the infection group, and the number of nodules in the viscera of the treatment group was also significantly lower than that of the infection group( Figure 9 ). This shows that the combined use of curcumin and quercetin can effectively improve the survival rate of large mouth bass after Nocardia seriolae infection.

[0104] III. Pathological observation and scoring

[0105] On the 8th day after challenge, randomly selected from four groups, respectively, large mouth bass, collection of its head kidney, spleen and liver and other tissues for H&E staining observation, and in accordance with the existing histopathology scoring system to make pathological score details. Pathological section making specific steps as follows:

[0106] (1) fixed: when the sample is fixed, use 500ml fixed bottle, take 1x1x0.5cm size of tissue, fixed in 4% neutral formalin solution or paraformaldehyde solution(the ratio of tissue and solution is about 1:9), 24-48h above.

[0107] (2) repair block: must be done in fume hood, pay attention to protection, fixed after repair tissue block, repair a flat surface, put into embedding box.

[0108] (3) decalcification: no bone tissue can be skipped, bone must be decalcified, put the bone and cartilage tissue sample into the decalcification solution for 24h, check the transparency; if not, continue to decalcify for 24h.

[0109] (4) water overnight and melt wax block: put the tissue into the water tank with embedding box, water overnight. When this step is performed, melt the wax block in advance in the 65℃ oven.

[0110] (5) dehydration embedding process:

[0111]

[0112]

[0113] (6) section: put into-20℃ refrigerator for more than 30min before sectioning, prepare ice box containing ice water mixture, take out the wax block and put it into the ice box, 10μm initial repair, after initial repair, ice box quick freezing, 4μm fine repair section, 40℃ or so spread the section, take out, dry.

[0114] (7) H&E staining observation.

[0115] The pathological observation and scoring results are as follows:

[0116] (1) head kidney histopathological changes and score

[0117] The head kidney of large mouth bass is characterized by no substantial renal units, which is mainly composed of reticular lymphoid tissue composed of lymphocytes and granulocytes. According to the arrangement characteristics of cells, it can be divided into two parts: granulocyte-rich area with light staining, and lymphocyte aggregation area stained with deep blue purple(H&E staining). Based on the above histological characteristics, the lesions of head kidney and their severity were scored systematically, and the specific scoring details are as follows:

[0118]

[0119] H&E staining revealed significant granulomatous lesions in the head kidney of the infected group, while no abnormalities were observed in the control group. The degree of lesions in the treatment group was significantly lower than that in the infected group. Figure 10 (See Figures A and B in the diagram). Specifically, the infected group showed extensive cell necrosis in both granulocyte and lymphocyte aggregation areas. Furthermore, the infected group exhibited significantly higher levels of granuloma coverage (granuloma diameter 0.19 ± 0.004 mm) and severity of caseous necrosis compared to the treatment group (granuloma diameter 0.06 ± 0.007 mm). Figure 10 (See diagrams C and D in the image). Further analysis of 1×1mm... 2 Quantitative analysis of the number of granulomas within the standard area showed that ( Figure 10 (Figure E in the diagram) The number of granulomas in the infected group was significantly higher than that in the treatment group (P<0.001). Based on the detailed pathological scoring criteria for head and kidney syndrome, it was found that ( Figure 10 (Figure F in the figure) The combined use of curcumin and quercetin can significantly reduce the degree of lesions in the head and kidney tissue (P<0.01).

[0120] (2) Pathological changes and scoring of spleen tissue

[0121] The spleen structure of the largemouth bass mainly consists of splenic cords and splenic sinuses. Specifically, the splenic cords are primarily composed of abundant lymphoid tissue, including lymphocytes, giant cells, plasma cells, erythrocytes, and granulocytes; the splenic sinuses are the main channels for blood flow between the splenic cords, mainly composed of flattened endothelial cells; in addition, the spleen is surrounded by a thin membrane. This invention scores the presence and severity of lesions in different locations, as follows:

[0122]

[0123] H&E staining analysis was performed on the spleen tissue of the largemouth bass from the four experimental groups. The results are as follows: Figure 11 As shown, the pathological damage to the spleen in the treatment group was significantly improved, with a significant decrease in the overall pathological score. Specifically, the number and diameter of granulomas were significantly reduced, the tissue structure was more regular, and the morphology of splenic sinuses was close to normal. In addition, the number of melanomacrophage centers (MMCs) in the treatment group was significantly increased. These results indicate that combined medication may combat infection by activating the immune system and enhancing the body's immune response. In conclusion, the combined use of curcumin and quercetin effectively alleviated spleen tissue damage caused by Nocardia auricula-judae infection.

[0124] (3) Liver histopathological changes and scoring

[0125] The liver tissue of the largemouth bass is mainly composed of two parts: one is the liver parenchyma composed of closely arranged hepatocytes, which is responsible for various metabolic functions; the other is the liver interstitium, which contains structures such as hepatic sinusoids, Disse's space and central veins, which jointly participate in material exchange and support blood circulation to provide necessary nutrients and oxygen for hepatocytes. The present application scores according to the presence or absence and severity of lesions in each part, and the specific scoring details are as follows:

[0126]

[0127] The observation of the liver tissue of the infected group of largemouth bass found that the main pathological changes were concentrated in the liver parenchyma and liver interstitium, and the tissue lesions of the treatment group were significantly improved. Specifically, the hepatocytes of the infected group showed obvious degeneration and necrosis, mainly manifested as enhanced cytoplasmic eosinophilia and karyopyknosis; at the same time, the hepatic sinusoids were dilated and accompanied by hyperemia, and the Disse's space was widened, indicating that there might be edema or inflammatory reaction locally. In contrast, the liver lesions of the treatment group were significantly reduced, and the number and area of granulomas were significantly reduced, and only a small amount of inflammatory cell infiltration was visible around the granulomas. In order to further evaluate the treatment effect, a comprehensive pathological score analysis was conducted on the two groups, and the results showed that the overall pathological score of the treatment group was significantly lower than that of the infected group, indicating that the combined use of curcumin and quercetin can also effectively improve the liver damage caused by Nocardia seriolae infection (for details, see Figure 12 ).

[0128] IV. Determination of serum non-specific immune enzyme activity

[0129] In order to evaluate the effect of combined medication on the serum enzyme activity of largemouth bass, 3 fish from each group were randomly selected and anesthetized with MS-222 and blood was collected from the tail vein on the 5th and 8th day after challenge. The collected blood was centrifuged at 8000 rpm for 10 min to obtain serum, which was stored at -20℃ for subsequent analysis. According to the instructions of the lysozyme (LYS), acid phosphatase (ACP), hydrogen peroxidase (H2O2) and superoxide dismutase (SOD) detection kits, the activities of enzymes in the serum samples of each group were determined, and three parallel samples were determined for each group. All data are expressed as mean ± standard deviation (Mean ± SD) to ensure the accuracy and reliability of the results.

[0130] The results showed that the infection of N. seriolae and the combined use of drugs significantly affected the activity of various enzymes in the serum, especially in the antioxidant stress response. Specifically, compared with the control group, the activities of lysozyme, acid phosphatase (ACP), superoxide dismutase (SOD) and catalase (CAT) in the serum were significantly increased (P<0.05) at 1d after administration (8d after infection), which was about 5 times that of the control group. Notably, although SOD and CAT returned to normal levels at 4d after administration (8d after infection), the activities of lysozyme and ACP remained at a high level, which may indicate that the body is still actively responding to infection or inflammation (see Figure 13 ).

[0131] V. Bacterial load determination of head kidney tissue

[0132] Based on the results of observation and histopathological scoring, combined with previous research findings, the bacterial load analysis of the head kidney tissue with the most severe lesions at 8d was conducted. The specific steps are as follows: select 3 largemouth bass from each group, cut the head kidney tissue and store it at -20℃. Before conducting the experiment, thaw the samples at room temperature, then add sterile phosphate buffer (PBS) at a ratio of 1:9, and grind thoroughly to prepare tissue homogenate. Next, take 20μL of the homogenate diluent and evenly spread the diluent on the culture medium (BHI) in a sterile operation table. Then, place the coated petri dish in a 28℃ incubator (24h), and finally count and statistically analyze the number of single bacteria on the petri dish.

[0133] The results showed that on the BHI solid medium, the number of bacteria in the head kidney tissue of the infected group (N.S) was significantly higher than that of the other groups, while the number of bacteria in the head kidney tissue of the treatment group (N.S+CQ) was significantly reduced (A-D graphs in Figure 14 ). In addition, the bacterial load of the head kidney tissue of the control group and the treatment group was significantly lower than that of the infected group (E graph in Figure 14 ). The above results show that the combined use of curcumin and quercetin can effectively reduce the bacterial load in the head kidney tissue of largemouth bass infected with N. seriolae.

[0134] VI. Determination of inflammatory factors in head kidney tissue

[0135] To explore the effect of the combined use of curcumin and quercetin on the expression of inflammatory factors in largemouth bass, the present invention analyzes the transcriptional level changes of key inflammatory-related genes (such as TNF-α, IL-1β, IL10, TGF-β2) in the head kidney tissue. The specific operation steps are as follows:

[0136] (1) RNA extraction and detection: On the 5th day of the challenge, 3 tails of all groups were randomly taken for sterile dissection, and the head kidney tissue was collected. Cell lysate was added to the collected tissue sample, and total RNA was extracted according to the tissue RNA extraction kit (Fuji Biological Technology Co., Ltd., Chengdu). The purity and concentration of the RNA were detected using a NanoDrop 200 spectrophotometer, and finally the integrity of the RNA was detected by agarose gel electrophoresis (1.5%).

[0137] (2) Synthesis of cDNA: The cDNA synthesis kit (Chengdu Fuji Biological Technology Co., Ltd., China) was used to synthesize cDNA after removing gDNA and reverse transcription in C1000TM Thermal Cycler PCR instrument, and finally the synthesized cDNA was stored at -80℃ for standby.

[0138] (3) Quantitative expression of genes: The primers used in the qRT-PCR experiment in the present application are designed based on the nucleotide sequence provided by the NCBI database (see the table below for details). Then, according to the operation manual of SYBR Premix Ex TaqTMII Perfect Real Time reagent kit, the StepOnePlusTM real-time fluorescent quantitative PCR system is used to complete the determination of the expression level of the target gene. The specific reaction system is as follows: upstream primer 0.4 μL (10 μM), downstream primer 0.4 μL (10 μM), SYBR Premix Ex TaqTMII (2×) 5 μL, ROX Reference Dye II 0.2 μL, ddH2O 3 μL, cDNA template 1 μL. The amplification program is set as follows: first 95℃ pre-denaturation for 3 min; then 40 cycles of amplification, including 95℃ denaturation for 10 s, specific annealing temperature extension for 30 s, and 72℃ final extension for 10 s. In order to ensure the accuracy of the results, the 18S rRNA was selected as the internal reference gene for calibrating the expression level of the target gene, and the 2 -△△Ct Method calculation of relative expression. Finally, the significance level of the expression difference between different samples was evaluated by Kruskal-Wallis H test, and P<0.05 was considered statistically significant, and P<0.001 indicated extremely significant difference.

[0139] The RT-qPCR primer sequences are as follows:

[0140] 18S-F: 5'-CGGCTACCACATCCAAGGAA-3'(SEQ ID NO.1);

[0141] 18S-R: 5'-CCTGTATTGTTATTTTTCGTCACTACCT-3'(SEQ ID NO.2);

[0142] TNF-a-F: 5'-CTTCGTCTACAGCCAGGCATCG-3' (SEQ ID NO. 3);

[0143] TNF-a-R: 5'-TTTGGCACACCGACCTCACC-3' (SEQ ID NO. 4);

[0144] IL-1 b-F: 5'-CGTGACTGACAGCAAAAAGAGG-3' (SEQ ID NO. 5);

[0145] IL-1 b-R: 5'-GATGCCCAGAGCCACAGTTC-3' (SEQ ID NO. 6);

[0146] IL10-F: 5'-CGGCACAGAAATCCCAGAGC-3' (SEQ ID NO. 7);

[0147] IL10-R: 5'-CAGCAGGCTCACAAAATAAACATC-3' (SEQ ID NO. 8);

[0148] TGF-b2-F: 5'-CGAAAATGCCATCCCACCA-3' (SEQ ID NO. 9);

[0149] TGF-b2-R: 5'-TGCCTTTGAATTCTGCAAACGA-3' (SEQ ID NO. 10).

[0150] The head kidney inflammation gene expression analysis showed that on the 5th day of challenge, the expression levels of pro-inflammatory factors IL-1 b and TNF-a were significantly up-regulated, about 20 times higher than those in the control group (Figures A and B in Figure 15 However, one day after administration, the expression levels of these two factors were significantly decreased; at the same time, the anti-inflammatory factors IL10 and TGF-b2 were also significantly up-regulated after infection, which might be related to the inhibition of excessive inflammatory response; one day after administration, their expression levels were decreased (Figures C and D in Figure 15 Figure 15 The above results showed that the combined administration not only reduced the formation of head kidney granuloma in a certain period of time, but also alleviated the excessive inflammation caused by infection.

[0151] ​The above merely describes preferred embodiments of the present application and is not used to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The application of a combination of curcumin and quercetin in the preparation of a treatment for nocardiosis in fish, a fish feed additive, or as a targeted inhibitor of the ICL receptor of Nocardia.

2. The application according to claim 1, characterized in that, Nocardiasis in fish is caused by Nocardia amberjack, Nocardia asteroides, or Nocardia salmonidae.

3. The application according to claim 1, characterized in that, The fish is a largemouth bass.

4. The application according to claim 1, characterized in that, In the combined use of curcumin and quercetin, the mass ratio of curcumin to quercetin is 1-5:1-5.

5. The application according to claim 4, characterized in that, In the combined use of curcumin and quercetin, the mass ratio of curcumin to quercetin is 1:

1.

6. The application according to claim 1, characterized in that, The dosage forms of drugs for treating nocardiosis in fish are oral preparations, soaking solutions, or injections.

7. The application according to claim 6, characterized in that, Treatments for nocardiosis in fish also contain pharmaceutically acceptable excipients.

8. The application according to claim 7, characterized in that, The excipients include at least one of a solubilizer, stabilizer, or aquatic feed binder.

9. The application according to claim 1, characterized in that, The minimum inhibitory concentration (MIC) of drugs for treating nocardiosis in fish is ≤4 μg / mL.

10. A target inhibitor of Nocardia ICL receptor, characterized in that, Including curcumin and / or quercetin.

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