Use of trimethylamine oxide in promoting liver and gill health in large-mouth bass

Abstract

The application discloses application of trimethylamine oxide in promoting liver and gill health of large-mouth bass, and explores the influence of trimethylamine oxide on liver and gill health of large-mouth bass by using trimethylamine oxide as a health regulator to feed large-mouth bass, including: tissue structure, cell apoptosis, glycogen content, serum ALT, p38MAPK activation, inflammatory response, 5-HT expression and the like. Research finds that trimethylamine oxide significantly relieves liver injury of large-mouth bass induced by high-sugar feed, and shows that liver glycogen accumulation and cell apoptosis are reduced, and serum ALT level is reduced; and trimethylamine oxide maintains gill tissue structure under ammonia nitrogen stress, inhibits p38MAPK signal path to weaken excessive inflammatory response, and reduces 5-HT in-situ expression to relieve excessive ventilation reaction induced by ammonia nitrogen. Therefore, trimethylamine oxide can be used as a health regulator to promote liver and gill health of large-mouth bass, and to enhance the resistance to high-sugar feed and ammonia nitrogen, so as to promote healthy aquaculture.

Description

Technical Field

[0001] This invention belongs to the field of fish health regulator technology, specifically relating to the application of trimethylamine oxide in promoting liver and gill health in largemouth bass. Background Technology

[0002] Largemouth bass (scientific name: *Micropterus salmoides*) belongs to the order Perciformes, family Heliotropidae, and genus *Micropterus*. With the continuous increase in aquaculture production, largemouth bass are experiencing frequent diseases, especially seriously threatening the health of tissues such as the liver and gills. In aquaculture production, the liver health of largemouth bass is significantly affected by the sugar level in the feed. Unlike other fish, excessively high sugar levels in the feed can cause glycogenic liver disease rather than fatty liver in largemouth bass. Furthermore, current largemouth bass farming is mainly based on intensive, high-density farming methods, leading to the continuous accumulation of ammonia nitrogen in the water, which severely damages the gill health of largemouth bass.

[0003] Trimethylamine oxide (TMAO) is a natural component found in various aquatic organisms. It can be produced by intestinal microorganisms such as betaine and choline, or by the catalysis of FMO3 in the liver. TMAO has a unique umami flavor and is a novel aquatic animal feed attractant. TMAO can also regulate osmotic pressure, stabilize protein structure, and function as a small molecule chaperone. Furthermore, TMAO can affect lipid absorption and cholesterol balance, and regulate glucose and lipid metabolism. Summary of the Invention

[0004] The purpose of this invention is to provide the application of trimethylamine oxide (TMO) in promoting liver and gill health in largemouth bass. Research has shown that TMO can alleviate liver damage induced by high-sugar diets in largemouth bass, reduce liver glycogen accumulation and glycogen-related liver disease, inhibit excessive hepatocyte apoptosis, and lower serum ALT levels. Furthermore, TMO can significantly maintain the normal structure of gill tissue under ammonia nitrogen stress, weaken the excessive inflammatory response induced by ammonia nitrogen stress by inhibiting the p38MAPK signaling pathway in the gills, and alleviate ammonia nitrogen-induced hyperventilation by reducing in situ expression of 5-HT. Therefore, it can be used as a health regulator to promote liver and gill health in largemouth bass and enhance their resistance to high-sugar diets and ammonia nitrogen in water, demonstrating high production application value.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides the application of trimethylamine oxide in promoting liver and gill health in largemouth bass.

[0006] Furthermore, the indicators of liver and gill health include: liver tissue structure, hepatocyte apoptosis, liver glycogen content, serum ALT level; gill tissue pathological analysis, p-p38MAPK protein expression, gill inflammatory response, and in situ expression of 5-HT protein in gill neuroepithelial cells.

[0007] Furthermore, the trimethylamine oxide promotes liver health in largemouth bass.

[0008] Furthermore, the trimethylamine oxide alleviates glycogen liver disease by reducing excessive glycogen accumulation in the liver of largemouth bass caused by high-sugar feed, inhibits excessive hepatocyte apoptosis, and reduces the level of ALT released into the serum, thereby promoting liver health in largemouth bass.

[0009] Furthermore, the trimethylamine oxide promotes gill health in largemouth bass.

[0010] Furthermore, the trimethylamine oxide inhibits the excessive activation of the p38MAPK pathway in the gills, thereby downregulating the expression of inflammatory cytokines il-1β and il-18 to suppress ammonia nitrogen-induced excessive inflammatory response in gill tissue, and alleviates ammonia nitrogen-induced hyperventilation response by reducing the in situ expression of 5-HT, thus maintaining the normal structure of gill tissue under ammonia nitrogen stress and promoting gill health in largemouth bass.

[0011] The present invention also provides a regulator of liver and gill health in largemouth bass, the regulator comprising trimethylamine oxide.

[0012] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention investigates the effects of trimethylamine oxide (TMO) on the liver and gill health of largemouth bass by feeding them with TME, including liver tissue structure, hepatocyte apoptosis, liver glycogen content, and serum ALT levels; gill tissue pathological structure, p-p38MAPK protein expression, gill inflammatory response, and in situ expression of 5-HT in gill neuroepithelial cells. The study found that TME significantly alleviates liver glycogen accumulation and glycogen-induced liver disease caused by high-sugar diets, inhibits excessive hepatocyte apoptosis, and reduces the level of ALT released into serum, thereby alleviating liver damage induced by high-sugar diets in largemouth bass. Furthermore, TME also reduces gill inflammatory responses induced by ammonia nitrogen stress by inhibiting excessive activation of the p38MAPK signaling pathway in the gills, and alleviates ammonia nitrogen-induced hyperventilation by reducing in situ expression of 5-HT in gill neuroepithelial cells, thus maintaining the normal structure of gill tissue under ammonia nitrogen stress and enhancing the resistance of largemouth bass to ammonia nitrogen stress. Therefore, trimethylamine oxide can be used as a health regulator to promote liver and gill health in largemouth bass and enhance its resistance to ammonia nitrogen in high-sugar feed and high-density aquaculture water. This is of great significance for promoting high-density intensive aquaculture of largemouth bass. Attached Figure Description

[0013] Figure 1 The results of HE and PAS staining of paraffin sections of liver tissue in Example 1 of this invention; Figure 2 This is the result of TUNEL staining to detect hepatocyte apoptosis in Example 1 of the present invention; Figure 3 The results of the detection of liver glycogen and serum ALT levels in Example 1 of this invention; Figure 4 HE staining results of paraffin sections of largemouth bass gill tissue before and after ammonia nitrogen stress in Example 1 of this invention; Figure 5 The results of Western blot analysis of p-p38MAPK in the gills in Example 1 of this invention; Figure 6 The results show the expression levels of the gill inflammatory cytokines il-1β and il-18 in Example 1 of this invention. Figure 7 This is the detection result of in situ expression of 5-HT protein in gill neuroepithelial cells in Example 1 of the present invention. Detailed Implementation

[0014] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] Example 1: Investigation into the effects of trimethylamine oxide on liver and gill health of largemouth bass 1. Test materials Healthy, uniformly sized largemouth bass (48.0 ± 2.0 g) were cultured in a recirculating aquaculture system at the experimental base of Huazhong Agricultural University. They were housed in 400L cylindrical tanks and divided into two groups: a control group and a TMAO group, with three tanks in each group and 15 fish per tank. Feeding was conducted twice daily, morning and evening. The control group was fed a largemouth bass diet containing 20% ​​carbohydrates, while the TMAO group was fed a diet containing 20% ​​carbohydrates plus an additional 0.25% (w / w) of TMAO. After eight weeks of continuous feeding, five fish were randomly selected from each tank for sampling. Dissolved oxygen was measured daily, and ammonia nitrogen and other water quality indicators were measured every other day.

[0016] 2. The regulatory effect of trimethylamine oxide on liver health of largemouth bass To investigate the alleviating effect of trimethylamine oxide treatment on liver damage induced by a high-sugar diet in largemouth bass, paraffin sections were stained with hematoxylin and eosin (HE) and PAS, hepatocyte apoptosis was detected by TUNEL assay, and liver glycogen content and serum ALT levels were measured. The specific experimental procedures are as follows: (1) Preparation of paraffin sections of liver tissue 1) Tissue fixation: Experimental fish were anesthetized with MS-222. Liver tissues from the control group and TMAO largemouth bass were taken and placed in 4% paraformaldehyde for more than 24 hours, ensuring that the fixative fully immersed the tissues.

[0017] 2) Gradient dehydration: The fixed tissue was sequentially transferred to 60% anhydrous ethanol for 4 h, 70% anhydrous ethanol overnight (8-10 h), 80% anhydrous ethanol for 2 h, 90% anhydrous ethanol for 2 h, 95% anhydrous ethanol I for 1.5 h, 95% anhydrous ethanol II for 1 h, 100% anhydrous ethanol I for 0.5 h, and 100% anhydrous ethanol II for 0.5 h.

[0018] 3) Transparency: Benzyl alcohol solution for 10 min, xylene I for 7 min, xylene II for 7 min.

[0019] 4) Waxing: Apply paraffin wax at 60℃ three times, changing the vat each time, and each time for 1 hour.

[0020] 5) Embedding: Place the paraffin-impregnated tissue into the center of the metal embedding frame and wait for the paraffin to solidify.

[0021] 6) Slicing: Trim the cut surface of the wax block and fix it on the microtome for slicing (thickness 5 µm). Transfer the slice to a spreader with water temperature of 41℃. Once the cut surface is flat, remove the slice and place it in a 65℃ constant temperature oven for 2 hours for later use.

[0022] (2) Hematoxylin-eosin (HE) staining procedure 1) Dewaxing: Remove the slices from the oven and then transfer them to xylene I for 20 min, xylene II for 15 min, and xylene III for 15 min to complete the dewaxing process.

[0023] 2) Displace xylene: Anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 75% anhydrous ethanol for 5 min, and wash with water for 3 min.

[0024] 3) Hematoxylin staining: Wash twice with water for 3 minutes (the time can be adjusted according to the effect of the hematoxylin staining solution), and then soak in water for 3 minutes.

[0025] 4) After washing, immerse the slide in the differentiation solution (99 mL 70% alcohol + 1 mL concentrated HCl) for 3-5 seconds, then rinse in water for 5 seconds, dry, and examine under a microscope. If the staining is too dark, continue rinsing in water for a period of time to fully remove HCl. If the differentiation is incomplete, it can be immersed in the differentiation solution again.

[0026] 5) After rinsing, immerse in the blueing solution (99 mL 70% alcohol + 1 mL concentrated ammonia) for 3-5 seconds, then rinse under distilled water and immerse in distilled water for 3 minutes.

[0027] 6) Immerse the slide in 85% ethanol for 5 min, 95% ethanol for 5 min, and eosin solution (1g of water-soluble eosin dissolved in 100 mL of 85% ethanol) for 2-3 min. If staining is difficult, add 1-2 drops of glacial acetic acid to every 100 mL of staining solution to make the tissue easier to stain and less prone to fading.

[0028] 7) Gradient alcohol dehydration: anhydrous ethanol I 5 min, anhydrous ethanol II 5 min, anhydrous ethanol III 5 min, n-butanol 5 min.

[0029] 8) Xylene I 5 min, Xylene II 5 min.

[0030] 9) Neutral resin sealing.

[0031] 10) Imaging: Images were acquired in the microscope (Olympus) using AXIOVISION software.

[0032] (3) Periodic acid Schiff (PAS) staining procedure 1) Dewaxing: Perform dewaxing as described above.

[0033] 2) Oxidation treatment: Treat the sections with an oxidant for 5-10 minutes, rinse with running water for 5 minutes, and wipe off excess water from the sections.

[0034] 3) Schiff staining: Add Schiff staining solution to the slide, stain for 10-15 min, and rinse with running water for 5-10 min.

[0035] 4) Perform hematoxylin counterstaining, differentiation, washing, blueing, and washing as described above.

[0036] 5) Dehydrate and clarify using the methods described above.

[0037] 6) Mount the specimen with neutral resin as described above and acquire images in a microscope (Olympus) using AXIOVISION software.

[0038] (4) TUNEL assay for hepatocyte apoptosis in largemouth bass; 1) Perform dewaxing and xylene displacement as described above; 2) Draw circles around the tissue with a histochemistry pen, and incubate with proteinase K diluted 1000 times with Tris-HCl at 37℃ for 20 min. Wash three times with PBS for 5 min each time.

[0039] 3) TUNEL assay solution preparation: 5 μL TdT enzyme + 45 μL fluorescent labeling solution for each sample. Prepare the required assay solution according to the number of samples.

[0040] 4) Incubate the TUNEL test solution at 37°C in the dark for 1 h.

[0041] 5) Wash 3 times with PBS, 5 min each time, and stain the cell nuclei with DAPI at room temperature for 8 min. 6) After washing twice with PBS, use absorbent paper to remove the water around the tissue, add anti-fluorescence attenuation mounting medium, and then mount with a coverslip. 7) Use an Olympus BX53 fluorescence microscope to acquire fluorescence signals, and use the corresponding imaging system to analyze and process the images.

[0042] (5) Liver glycogen content detection 1) Weigh 0.1 g of sample and place it in a 10 mL test tube; add 0.75 mL of extraction solution, place in a boiling water bath and boil for 20 min (tightly cover to prevent moisture loss), shake the test tube once every 5 min to mix thoroughly; after the tissue has completely dissolved, remove the test tube and cool it, then dilute to 5 mL with distilled water, mix well, centrifuge at 8000 g at 25℃ for 10 min, and take the supernatant for testing.

[0043] 2) Preheat the microplate reader for at least 30 minutes and adjust the wavelength to 620 nm.

[0044] 3) Reagent dilution: Take 10 μL of 10 mg / mL glucose standard solution, add 990 μL of distilled water, mix thoroughly, and prepare a 0.1 mg / mL glucose solution for use only.

[0045] 4) Add samples according to the table below.

[0046] Table 1. Sample Addition Order for Glycogen Testing 5) Mix well, boil in a water bath for 10 min (tightly cover to prevent moisture loss), cool, take 200 μL and transfer to a 96-well plate, and read the absorbance of the blank tube, standard tube and test tube at a wavelength of 620 nm, and record them as A1, A2 and A3 respectively.

[0047] 6) Calculation of glycogen content: Glycogen (mg / g mass) = (C standard × V1) × (A3 - A1) ÷ (W × V1 ÷ V2) ÷ 1.11 = 0.450 × (A3 - A1) ÷ (A2 - A1) ÷ W (sample mass) (6) Serum ALT level detection 1) Dilution of the standard curve: First, dilute the standard (20 μmol / mL sodium pyruvate) with distilled water to 2 μmol / mL as the standard diluent. Then, mix the standard diluent with reagent one to obtain concentration gradient standard tubes with concentrations of 1, 0.8, 0.4, 0.2, 0.1, 0.05, and 0 μmol / mL.

[0048] 2) Thaw the serum sample in ice for later use.

[0049] 3) Add reagents according to Table 2.

[0050] Table 2 ALT activity detection system 4) Mix well, let stand at room temperature for 10 min, and measure the absorbance of each tube at a wavelength of 505 nm.

[0051] 5) Plotting the standard curve: Plot the standard curve with the concentration of each standard solution as the x-axis and ΔA (A standard tube - A blank tube) as the y-axis, obtaining the equation y = kx + b. Substitute (A test tube - A control tube) into the equation to find the value of x (μmol / mL).

[0052] 6) Calculation of ALT activity: One unit of ALT activity is defined as the amount of pyruvate produced per mL of serum sample per hour. ALT activity (U / mL) = x × (Vsample + Vreagent) ÷ T = 12x (7) Results and Analysis Test results as follows Figure 1 , Figure 2 and Figure 3 As shown, where Figure 1 -(a) HE staining images of paraffin sections of liver tissue from the control group and the TMAO group (scale bar: 50 µm). Figure 1 -(b) shows the PAS staining pattern of a paraffin section of liver tissue; Figure 2 The results of liver TUNEL assays are for the control group and the TMAO group. Figure 3 -(a) shows the results of liver glycogen content detection. Figure 3 -(b) shows the results of serum ALT level detection.

[0053] Figure 1HE staining results in (a) showed that in the control group, high sugar diet caused damage to the cell membrane of largemouth bass hepatocytes, and the cell nuclei were irregularly distributed and mostly aggregated to the periphery. In contrast, in the TMAO group, hepatocytes were arranged radially around the central vein to form cell plates, and the related structures were intact, indicating that TMAO significantly alleviated liver damage induced by high sugar diet. Figure 1 -(b) PAS staining results and Figure 3 -(a) The glycogen content detection results showed that in the control group, the high sugar diet led to a significant signal of glycogen accumulation in the liver cells of largemouth bass, with a glycogen content of up to 184 mg / g tissue. This proved that the high sugar diet led to glycogen accumulation in the liver of largemouth bass and induced glycogen liver disease. In contrast, the glycogen signal in the liver cells of the TMAO group was significantly reduced, and the liver glycogen content was significantly lower than that of the control group. This indicated that TMAO significantly alleviated the glycogen accumulation and glycogen liver disease induced by the high sugar diet in largemouth bass. Figure 2 TUNEL assay results showed that the high-sugar diet in the control group induced significant TUNEL expression in hepatocytes. + Signal, and TUNEL in TMAO group + The signal was significantly lower than that in the control group, indicating that TMAO inhibited excessive hepatocyte apoptosis induced by high-sugar diet; Figure 3 The serum ALT level detection results in (b) showed that the serum ALT level in the TMAO group was significantly lower than that in the control group due to liver damage caused by high-sugar diet, indicating that TMAO significantly alleviated liver damage and serum ALT levels induced by high-sugar diet.

[0054] 3. The regulatory effect of trimethylamine oxide on gill health of largemouth bass To investigate the alleviating effect of trimethylamine oxide treatment on ammonia nitrogen-induced gill damage in largemouth bass, gill tissue pathology was performed using paraffin sectioning and HE staining. Western blotting was used to detect the activation level of the p38MAPK signaling pathway. Real-time quantitative PCR was used to detect the gene expression of inflammatory cytokines il-1β and il-18 in gill tissue. Immunofluorescence was used to detect the in situ expression of 5-HT protein in gill neuroepithelial cells. The specific experimental steps are as follows: (1) The gill tissue morphology was observed by HE staining according to the aforementioned method; (2) Western blot analysis to detect the activation level of the p38MAPK signaling pathway in gills; 1) Protein Sample Preparation: Weigh 150 mg of frozen gill tissue into a pre-chilled glass homogenizer. Add 1.5 mL of RIPA composite lysis buffer to the homogenizer and homogenize on ice until no obvious tissue fragments are visible. Transfer to a new centrifuge tube. Place the centrifuge tube on a four-dimensional mixer in a 4 ℃ chromatography cabinet and mix for 30 min to ensure thorough mixing of the tissue fragments and lysis buffer, achieving complete protein lysis. Centrifuge at 12000 r / min for 20 min at 4 ℃, and collect the supernatant and place it in an ice-water bath. Subsequently, use the BCA protein concentration assay kit from Beyotime International Co., Ltd. to determine the protein concentration. After adjusting the protein content of the two groups to be consistent, aliquots and store.

[0055] 2) SDS-PAGE electrophoresis: The balanced sample protein extract was added to 5× protein loading buffer (reducing) at a ratio of 1:4 to prepare the loading protein solution. After slight mixing, the mixture was heated to boiling for 10 min. Then, the sample was centrifuged at 12000 rpm for 1 min at room temperature to avoid the presence of possible impurities. The supernatant was transferred to a new centrifuge tube. Subsequently, the loading protein solution was added to each well, with 20 µL of loading protein solution added to each well. The first well was filled with 5 µL of pre-stained standard molecular weight protein. The gel plate with the loaded sample was placed on an electrophoresis apparatus under constant voltage (80V) for vertical electrophoresis for 120 min, and the electrophoresis was stopped when the bromophenol blue label reached the bottom of the gel plate.

[0056] 3) Transfer to PVDF membrane: Place the electrophoresed gel in the transfer buffer for 1 min to equilibrate and remove the bound SDS on the membrane. At the same time, cut a PVDF membrane of appropriate size and soak it in methanol for 15 s to activate the PVDF membrane. Then, immerse the membrane and filter paper in the transfer buffer for 3 min and arrange them from bottom to top in the order of filter paper-PVDF membrane-gel-filter paper, ensuring that there are no air bubbles in each layer. Finally, gently press with a roller, add some more transfer buffer, and finally cover the instrument. Transfer the membrane for 50 min under constant current (0.4 mA for one membrane, 0.8 mA for two membranes).

[0057] 4) Blocking: Prepare a 5% skim milk powder blocking solution, and wash the PVDF membrane with PBST for 5 min after the transfer. Then, immerse it face up in the 5% skim milk powder solution and gently shake it on a shaker at room temperature for 2 h to block non-specific binding sites. After blocking, rinse the PVDF membrane once with PBS solution.

[0058] 5) Incubation with primary antibody: Dilute the primary antibody against rabbit anti-p-p38MAPK at a ratio of 1:1000 in PBST buffer according to the instructions, then add it to the PVDF membrane containing the protein sample, incubate at room temperature for 1 h, and wash with PBST 4 times for 5 min each time.

[0059] 6) Second antibody incubation: Add the prepared goat anti-rabbit second antibody, incubate at room temperature for 45 min, wash with PBST 3 times, 5 min each time; finally wash with PBS for 5 minutes.

[0060] 7) ECL development: Add ECL development solution and use a chemiluminescence imaging system for imaging. Adjust the parameters until the bands are clear.

[0061] (3) Real-time quantitative PCR was used to detect the expression of the gill inflammatory cytokines il-1β and il-18 genes; 1) RNA Extraction: Remove tissue samples from the -80 ℃ freezer and place them in a 2 mL RNAiso Plus centrifuge tube containing 1 mL TRIzol reagent. Add DEPC-sterilized glass beads and homogenize in a homogenizer for 1 min, observing whether the tissue in the TRIzol solution is completely broken down. Immediately incubate at 4 ℃ for 10 min. Add 0.2 mL chloroform, vigorously shake the centrifuge tube for 10 s, incubate at 4 ℃ for 5 min, and centrifuge at 12000 r / min at 4 ℃ for 15 min. Carefully transfer the supernatant to a new 1.5 mL RNAiso Plus centrifuge tube, add an equal volume of isopropanol, gently invert the tube several times to ensure the isopropanol reacts fully with the supernatant, incubate at 4 ℃ for 10 min, and centrifuge at 12000 r / min at 4 ℃ for 10 min. Discard the supernatant, add 1 mL of 75% ethanol prepared with DEPC water, re-vortex the precipitate, and centrifuge at 7500 r / min at 4 ℃ for 5 min. min; discard the supernatant, repeat the above steps, and then place on ice in a fume hood to dry for about 10 min until the 75% ethanol in the centrifuge tube has completely evaporated. Add an appropriate amount of DEPC water to dissolve the precipitate. If necessary, gently blow the precipitate with a pipette to dissolve it completely. Use a NanoDrop spectrophotometer to detect the purity and concentration of RNA, and use 1% agarose gel electrophoresis to detect RNA integrity.

[0062] 2) cDNA synthesis: First, genomic DNA was removed by treating the 5*g DNA digester mix from the Yeasen reverse transcription kit at 42℃ for 2 min in a PCR instrument. Then, 5μL of 4× Hifair®Ⅲ SuperMix Plus was added to the 15μL reaction solution. After adding reagents sequentially, the mixture was vortexed and centrifuged, then transferred to a PCR instrument for reaction. The reaction conditions were: 25℃ for 10 min, 42℃ for 90 min, and 85℃ for 5 min. The obtained cDNA was diluted to the same concentration with an appropriate amount of deionized water and stored at -20℃.

[0063] 3) Real-time quantitative PCR: Add 1.0 μL of cDNA template (200 ng / μL) and 0.15 μL each of upstream and downstream primers to 5.0 μL of Hifair® qPCR SYBR Green Master Mix reagent, and bring the volume to 10 μL with deionized water. Mix the reaction mixture thoroughly and transfer it to a light-protected 96-well plate. Seal the plate and centrifuge at low speed for 10 seconds. Then, use qTOWER... 3 qRT-PCR was performed using a G-type real-time PCR instrument under the following conditions: 95 °C pre-denaturation for 5 min, 95 °C denaturation for 10 s, and 57 °C extension for 30 s, for a total of 40 cycles. Melting curves were included, and each sample was tested in triplicate. After qRT-PCR, melting curves were analyzed to ensure reaction specificity. The 18S gene was used as an internal reference. Relative quantitative data were analyzed using a 2-1TLC method. -ΔΔCt Methods. qRT-PCR data were analyzed using GraphPad Prism 6. Data represent at least three independent experiments (mean ± standard error). Student's t-test was used for data analysis. P < 0.05 was indicated by *, representing statistical significance; P < 0.01 was indicated by **; and P < 0.001 was indicated by ***, all representing highly statistical differences.

[0064] (4) Immunofluorescence detection of in situ expression of 5-HT protein in gill neuroepithelial cells; 1) The paraffin sections obtained above are dewaxed and dexylene-removed; 2) Repair the above slides with 1% EDTA-2Na repair solution in a microwave oven, and after they have cooled to room temperature, wash the slides twice with PBS, 5 min each time. 3) Draw circles around the tissue with a histochemistry pen, block with protein blocking solution for 30 minutes, and rinse with PBS to remove the blocking solution; 4) Add diluted rabbit 5-HT antibody to the tissue sections, incubate overnight at 4ºC, and wash 4 times with PBS to remove unbound antibody; 5) Add diluted goat anti-rabbit secondary antibody to the tissue sections, incubate at room temperature for 45 min, and wash 4 times with PBS to remove unbound antibody; 6) Use DAPI to stain the cell nuclei at room temperature for 8 min. After washing twice with PBS, use absorbent paper to remove water from the tissue, add anti-fluorescence attenuation mounting medium, and then mount with coverslip. 7) Use an Olympus BX53 fluorescence microscope to acquire fluorescence signals, and use the corresponding imaging system to analyze and process the images.

[0065] (5) Results and Analysis Test results as follows Figure 4 , Figure 5 , Figure 6 and Figure 7 As shown, where Figure 4 HE staining images of gill tissue sections of largemouth bass in the control group and TMAO group before and after ammonia nitrogen stress (scale bar: 50 µm). Figure 5 The image shows the protein blot results of p-p38MAPK in the gills of largemouth bass after ammonia nitrogen stress. Figure 6 The results show the gene expression of il-1β and il-18 inflammatory cytokines in the gill tissue of largemouth bass in the control group and TMAO group after ammonia nitrogen stress. Figure 7 This study describes the in situ expression of 5-HT protein in the gill neuroepithelial cells of largemouth bass before and after ammonia nitrogen stress.

[0066] Figure 4 HE staining results showed that the gill tissue of the control group of largemouth bass showed obvious lesions after ammonia nitrogen stress, with shortened secondary gill filaments. In contrast, the length of secondary gill filaments in the gills of the TMAO group of largemouth bass after ammonia nitrogen stress was significantly greater than that in the control group. The above results indicate that TMAO maintained the structural integrity of the gill tissue of largemouth bass after ammonia nitrogen stress. Figure 5 Western blot results showed that the p-p38MAPK activation level in the gills of the control group was very high after ammonia nitrogen stress, while TMAO significantly inhibited the excessive activation level of the p38MAPK pathway in the gills of the largemouth bass induced by ammonia nitrogen. Figure 6 Real-time quantitative PCR showed that TMAO significantly inhibited the gene expression levels of inflammatory cytokines il-1β and il-18 in the gills of largemouth bass induced by ammonia nitrogen. Figure 7 Immunofluorescence results showed that 5-HT expression was significantly increased in the gill neuroepithelial cells of the control group after ammonia nitrogen stress, while 5-HT expression in the gill neuroepithelial cells of the TMAO group was significantly lower than that in the control group after ammonia nitrogen stress. These results indicate that TMAO inhibits the 5-HT-mediated hyperventilation response in the gill neuroepithelial cells of largemouth bass induced by ammonia nitrogen stress, thereby maintaining the integrity of gill tissue structure and function.

[0067] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of trimethylamine oxide in promoting liver and gill health in largemouth bass, characterized in that, Supplementing a 20% carbohydrate diet containing trimethylamine oxide with 0.25% by mass of the feed used to feed largemouth bass twice daily for 8 weeks; the promotion of liver health includes reducing liver glycogen content and serum alanine aminotransferase levels, and the promotion of gill health includes alleviating gill tissue damage following ammonia nitrogen stress.

2. The application according to claim 1, characterized in that, The trimethylamine oxide inhibits excessive apoptosis of largemouth bass hepatocytes under high-sugar diet conditions.

3. The application according to claim 1, characterized in that, The trimethylamine oxide reduced the gene expression levels of il-1β and il-18 inflammatory factors in the gill tissue of largemouth bass after ammonia nitrogen stress.

4. The application according to claim 1, characterized in that, The trimethylamine oxide reduced the in situ expression of 5-hydroxytryptamine protein in the gill neuroepithelial cells of largemouth bass after ammonia nitrogen stress.

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