Application of quercetin in aquaculture

Abstract

The technical scheme provided by the invention is application of quercetin in aquaculture, in particular to application of quercetin in preparation of drugs and / or feeds for preventing and treating thyroxine disorder and / or serotonin disorder of aquatic animals. The invention has the beneficial technical effects that the natural plant component quercetin which is originally applied to medicines and health care products for people is expanded to be applied to the field of aquatic animals, so that the nutrition required by the aquatic animals can be provided, the effect of regulating thyroid hormone and / or serotonin disorder of the aquatic animals can be achieved, the quercetin is used for replacing hormone medicines, and the health care effect is good. The method is friendly to an ecological chain, and green, cyclic and sustainable development is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of aquaculture technology, in particular, the application of quercetin in aquaculture. BACKGROUND

[0002] As a natural polyhydroxy flavonoid, quercetin has shown wide application value in various fields due to its unique biological activity. In the field of medicine, quercetin shows significant efficacy in treating various malignant tumors such as cervical cancer, gastric cancer, and liver cancer through inhibiting tumor cell proliferation, inducing apoptosis, and blocking metastasis. Its antioxidant capacity is 50 times that of vitamin E, which can effectively scavenge free radicals and protect nerve cells from oxidative damage, and has potential in the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease. In addition, quercetin can also help treat diabetes and hyperlipidemia by regulating glucose and lipid metabolism, inhibiting alpha-amylase activity and cholesterol absorption. Its ability to inhibit xanthine oxidase makes it useful in the management of hyperuricemia and gout. For example, the patent document with publication number CN116650472A and the title "Application of Quercetin" discloses the use of quercetin as a drug for treating intestinal damage.

[0003] In the food industry, quercetin is often used as a natural antioxidant to delay oil oxidation and spoilage, prolong the shelf life of meat products, beverages, and cold drinks. At the same time, its polyphenol structure can enhance the light stability of food pigments and maintain product color. It is worth noting that quercetin is gradually gaining attention in the development of functional foods and is being made into dietary supplements to meet the needs of antioxidant and anti-inflammatory health. However, its low water solubility and fast metabolism limit its bioavailability, and improving its purity and stability through enzymatic conversion and other technologies is an important direction for expanding its applications. Currently, the application of quercetin is extending from traditional medicine to food and health products, and its development potential in precision medicine and natural health products is promising.

[0004] In summary, quercetin, as a natural plant-derived active ingredient, has formed a mature technical system in the fields of medicine and food. If its application field is expanded to the field of aquaculture technology, especially for the disease problems that easily occur in the process of aquaculture, it not only breaks through the technical bottleneck of traditional antibiotic dependence, but also constructs a green ecological breeding mode. The development of water production special products based on quercetin has important research value and industrialization prospects for promoting the sustainable development of aquaculture. SUMMARY

[0005] The present application aims to break through the existing application boundaries of quercetin and build a multi-dimensional application system in the field of aquaculture.

[0006] In order to achieve the above-mentioned purpose, the technical scheme provided by the present application is the application of quercetin as a drug and / or feed for preventing and treating thyroid hormone disorder and / or serotonin disorder of aquatic animals. Quercetin is currently more commonly used in the field of human drugs and health foods, and is mostly used in anti-aging, auxiliary treatment of diabetes, and regulation of lipid metabolism. The present application innovatively finds that quercetin can also intervene in the regulation of hormone disorders of aquatic animals. In the prior art, aquatic animals mostly add iodine elements in feed to intervene in thyroid hormone disorders by supplementing iodine. In addition to supplementing iodine, hormone drugs are also commonly used for treatment, such as levothyroxine and propylthiouracil. For serotonin disorders in the prior art, hormone drugs such as fluoxetine and sertraline are commonly used for regulation. The present application uses natural plant-derived ingredients, which are easy to degrade and not easy to remain, and is friendly to the ecological chain. Therefore, more and more natural plant-derived ingredients are developed to provide necessary nutrients for aquatic animals while intervening in the diseases of aquatic animals, and to enable the aquatic animal breeding industry through technical empowerment, thereby improving the quality and efficiency of the aquatic animal breeding industry.

[0007] Specifically, the aquatic animal is rare gobi-Przewalskia. The present application finds that quercetin is most effective for thyroid hormone disorder and / or serotonin disorder of rare gobi-Przewalskia.

[0008] Preferably, the mass concentration of quercetin is 1-100 μg / L. Quercetin is generally dissolved in ethanol or DMSO. In this concentration range, the thyroid hormone disorder and / or serotonin disorder of gobi-Przewalskia can be well regulated while providing nutrition, thereby optimizing the cost and effect.

[0009] Preferably, when quercetin is used for preparing a drug for preventing and treating thyroid hormone disorder and / or serotonin disorder of aquatic animals, the drug further comprises a drug lipid carrier acceptable to quercetin. The lipid carrier is composed of apolipoprotein A-1 and lecithin, and the mass ratio of apolipoprotein A-1 to lecithin is 1-5:2-6. The mass ratio of the lipid carrier to quercetin is 30-50:20-50. In order to enhance the bioavailability of quercetin, the present application selects the lipid carrier for delivery of quercetin.

[0010] Further, the preparation method of the quercetin wrapped lipid carrier adopts microfluidic preparation. The method comprises the following steps: (1) preparing an inorganic phase: adding apolipoprotein A-1 single analog peptide to an inorganic reagent to obtain solution A; (2) preparing an organic phase solution: adding quercetin and lecithin to an organic reagent to obtain solution B; (3) mixing solution A and solution B by microfluidic to obtain the drug.

[0011] The apolipoprotein A-1 single simulation peptide in the step (1) is dissolved with PBS, and the mass: volume of the apolipoprotein A-1 single simulation peptide and PBS is 1-5 mg: 10 ml; The organic reagent in the step (2) is ethanol, and the mass: mass: volume of quercetin, lecithin and ethanol is 0.1-0.5 mg: 2-8 mg: 5-10 ml.

[0012] The microfluidic method in the step (3) is a microfluidic method for synthesizing a mixture of quercetin wrapped lipid carriers according to an organic phase 1 mL / min-5 mL / min flow rate and an inorganic phase 5 mL / min-10 mL / min flow rate.

[0013] Therefore, the natural plant component quercetin originally applied to human medicine and health products is expanded to the field of aquatic animals, which not only provides the nutrients required by aquatic animals, but also plays a role in regulating thyroid hormone and / or serotonin disorders of aquatic animals, and is used to replace hormone drugs, is friendly to the ecological chain, and realizes green and sustainable development. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 For the effects of MT and different doses of quercetin on the thyroid tissue of Gobiocypris rarus (400x, scale 50um), in the figure, colloid (c), thyroid follicle cells (f), colloid loss (cd), thyroid follicle cell hyperplasia (ft), disordered arrangement ( ); Figure 2 For the thyroid follicle epithelial cell hyperplasia rate and the enlargement rate; Figure 3 Effects of MT and different doses of quercetin on thyroid hormone-related genes of Gobiocypris rarus; Figure 4 Effects of MT and different doses of quercetin on serotonin-related genes of Gobiocypris rarus; Figure 5 Effects of MT and different doses of quercetin on the content of thyroid and serotonin of Gobiocypris rarus; Figure 6 Wayne diagram of differentially expressed genes of Gobiocypris rarus treated with MT and 10ug / L quercetin alone or together; Figure 7 Volcano plot of differentially expressed genes of Gobiocypris rarus treated with MT and 10ug / L quercetin; Figure 8 GO enrichment of differentially expressed genes of Gobiocypris rarus treated with MT and 10ug / L quercetin; Figure 9 KEGG enrichment of differentially expressed genes of Gobiocypris rarus treated with MT and 10ug / L quercetin.

[0015] The above figure Figures 1-5 In the above figure, each group is: the CON column is the control group; the MT column is the 50 ng / L MT group; the MT+1Q column is the 50 ng / L MT + 1ug / L quercetin group; the MT+10Q column is the 50 ng / L MT + 1ug / L quercetin group; and the MT+100Q column is the 50 ng / L MT + 100 ug / L quercetin group. DETAILED DESCRIPTION

[0016] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0017] Embodiment one: The present application finds that quercetin can be used for preparing a medicine for preventing and treating thyroid hormone disorder and / or serotonin disorder in aquatic animals. The following takes rare gobiocypris as an example to illustrate that quercetin can regulate thyroid hormone disorder and serotonin disorder.

[0018] I. Experimental content 1. Exposure of rare gobiocypris and sample collection The fish disposal in the present study was in accordance with the Animal Protection Ethics Committee of Shanxi Agricultural University (No. SXAU-EAW-2022F.VW.007026001). The 5-month-old rare gobiocypris used in the present study were obtained from the Aquaculture Laboratory of Shanxi Agricultural University. Before the start of the experiment, these fish were acclimated for 1 week in a fully ventilated and oxygenated aquarium. After acclimation, 250 rare gobiocypris were randomly divided into 5 groups, namely 0.01% DMSO, 50 ng / L MT, 50 ng / L MT and 1ug / L quercetin, 50 ng / L MT and 10ug / L quercetin, and 50 ng / L MT and 100ug / L quercetin. The doses of DMSO, quercetin and MT were selected according to previous studies. The water used in the present experiment was tap water, which was exposed to aeration and semi-sterile water for 24 h, with a pH range of 7.6±0.2 and a temperature range of 25±1℃; the light cycle was artificially controlled at 14h: 10h light: dark. The residual food and excrement were pumped out daily, and 1 / 2 of the water was replaced at the same concentration. The fish were fed twice a day, with a bloodworm at 1.5% of the total weight of the test group.

[0019] After 28 d, 25 G. aequus were randomly selected from each group. G. aequus were anesthetized in an ice water mixture and immediately dissected to collect the head and intestine for further analysis. Five heads and five intestines were fixed in Bouin’s solution for histopathological analysis. Three heads were frozen at -80 °C for hormone level determination. The remaining samples were quickly frozen in liquid nitrogen and stored at -80 °C for subsequent analysis.

[0020] 2. Histopathological observation of thyroid After 24 h fixation, samples (n = 5) were rinsed with running water for 24 h, dehydrated with graded ethanol, dehydrated with xylene, and then embedded in paraffin. Subsequently, the specimens were cut into 5 μm using a rotary microtome (paraffin machine, Leica RM2245, Germany). After hematoxylin-eosin staining and neutral resin sealing, the percentage of thyroid follicular epithelial cell nuclear hypertrophy and the number of hyperplasia per section were measured using Motic Imagic Plus software. The number of follicular epithelial cells was counted within an area (1.19 mm x 8.97 mm), and then the percentage and specified number were estimated.

[0021] 3. Total brain RNA extraction and real-time PCR Total RNA of each group of 3 Gobiocypris rarus was extracted by using Trizol (TaKaRa, Japan) one-step method. Then the quality of RNA was verified by measuring the ratio of 260 / 280 nm with spectrophotometer (M2, Molecular Devices, Sunnyvale, CA, USA). Reverse transcription of cDNA was performed using PrimeScript™ RT Master Mix (Takara Biomedical Technology Co. Ltd, Beijing, China). TB Green™ Premix Ex Taq™ II kit (Takara Biomedical Technology Co. Ltd, Beijing, China) and QT system (Thermo, USA) were used to detect mRNA expression. The final volume of the reaction was 20 μL. The reaction conditions were: pre-denaturation at 95 °C for 10 min, denaturation at 95 °C for 15 s, annealing / extension at 60 °C for 1 min, 41-45 cycles. The specificity of the amplified product was verified by melting curve analysis at the end of real-time fluorescence quantitative PCR (qPCR). Specific primers were for corticotropin-releasing hormone (crh), tryptophan hydroxylase (tph), tryptophan hydroxylase (tph1), deiodinase 1 (dio1), deiodinase 2 (dio2), sodium / iodide symporter (nis), udp-glucuronosyltransferase (ugt1a), thyroid hormone receptor alpha (tr-a), thyroid peroxidase (tpo), thyroid hormone receptor beta (tr-b), serotonin receptor 1Aa (htr1aa), serotonin receptor 1Ab (htr1ab), serotonin receptor 1d (htr1d), and β-actin (housekeeping gene). Primer Premier 5 was used to design the related genes, and Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. synthesized them. The detailed information is shown in Table 1. The transcriptional expression level of these genes was normalized to the transcriptional expression level of β-actin using the 2-ΔΔCt method.

[0022]

[0023]

[0024] 4. Measurement of TS, T3, T4 levels in the brain The head tissue (n = 3) was placed in 4 °C PBS (pH = 7.4, w / v = 1:9) and homogenized in an ice bath to prepare 10% homogenate. After centrifugation at 4 °C (3000 rpm, 20 min), the supernatant was collected, The purified fish serotonin (ST), tetraiodothyronine (T4), triiodothyronine (T3) antibody-coated microplate was made into solid phase antibody, and the serotonin (ST), tetraiodothyronine (T4), triiodothyronine (T3) was added into the microplate coated with monoclonal antibody in turn, and then combined with the corresponding hormone antibody labeled with HRP to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB was added for color development. TMB is converted to blue under the catalysis of HRP enzyme, and is converted to the final yellow under the action of acid. The color depth is positively correlated with the ST, T4 and T3 in the sample. The absorbance (OD value) was measured at 450 nm wavelength by enzyme-labeled instrument, and the fish TS, T4 and T3 concentrations in the sample were calculated by standard curve, and the detection limits were 7.34 pmol / L, 60 pmol / L and 1 pmol / L, respectively.

[0025] 5.16S rRNA gene amplification and high-throughput sequencing Based on the results of histology, real-time fluorescence quantification, and Elisa, we selected the intestinal microbiota samples of CON, MT, and MT+10Q groups for analysis. Genomic DNA was extracted from the intestinal content samples according to the instructions of the commercial HiPure Stool DNA Kits (Magen, Guangzhou, China). The integrity of the extracted DNA was detected by 1% agarose gel electrophoresis. The 16S rRNA gene sequences were analyzed using bacterial 16S rRNA gene V3 to V4 region primers 341F (5'-CCTACGGGNGGCWGCAG-3') and 806R (5'-GGACTACHVGGGTATCTAAT-3'), and the operational taxonomic units (OTUs) were used for analysis, with a threshold of 97% pair-wise identity, and the Ribosomal Database Project (RDP) classifier 2.0.1 was used for classification.

[0026] 6. Brain tissue transcriptome sequencing The same as above, samples from CON, MT, MT+10Q groups were selected for analysis. At the end of the 28-day experimental period, the whole brain of G. a. deu was extracted, and each treatment contained 3 parallel replicates. Total RNA was extracted from brain tissue using TRIzol reagent (Life Technologies, Carlsbad, CA, USA). RNA was detected by Agilent 2100 Bioanalyzer (Agilent Technologies, USA), and then commercial RNA-seq analysis was performed using Illumina HiSeq2500 of Gene Denovo biotech Co. (Guangzhou, China). The raw data were filtered by fastp (version 0.18.0) to obtain high-quality and reliable pure reads. Paired clean reads were mapped using HISAT2.2.4. The expression abundance and changes of each gene were normalized to the number of transcripts per million (TPM) using the RNA-seq by Expectation Maximization (RSEM) method. The differentially expressed genes (DEGs) with |log 2 (fold change)|≥1.8 and p-adjust <0.05 were further analyzed by GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of genes and Genomes). The above analysis steps were performed on the GENE DENOVO (http: / / www.omicsmart.com) online platform.

[0027] 7. Statistical analysis The data were expressed as mean ± SEM (standard error), and when the data met the assumption of homogeneity of variance (test value = 0.05), one-way ANOVA was used to analyze the data by SPSS19.0. If there was a significant difference between the control group and the treatment group, Duncan's method was used for multiple comparisons. p<0.05 was considered statistically significant.

[0028] II. Results 2.1 Effect of quercetin on thyroid tissue exposed to MT The histological results of thyroid showed that the CON group had complete thyroid follicles of different sizes, containing colloid, with single-layer epithelial cells arranged in order, small and round nuclei, and light staining. The MT group showed thyroid follicular epithelial hyperplasia, large nuclei, deep staining, blurred structure between follicular epithelial cells, disordered arrangement, and colloid loss. In the treatment group, the MT+1Q and MT+10Q groups showed reduced follicular epithelial cell hyperplasia, clear boundaries between follicular epithelial cells, relatively orderly arrangement, and reduced cell nucleus swelling. The MT+100Q group still showed obvious hyperplasia, colloid loss, blurred boundaries between follicular epithelial cells, and disordered arrangement. Figure 1 .

[0029] The results of thyroid follicular epithelial cell hyperplasia rate showed that compared with the MT group, the MT+1Q group decreased by 40%, the MT+10Q group decreased by 60%, and the MT+100Q group decreased by 60%. The results of thyroid follicular epithelial cell swelling incidence showed that compared with the MT group, the MT+1Q group decreased by 75.97%, the MT+10Q group decreased by 80.33%, and the MT+100Q group decreased by 47.82% ( Figure 2 ).

[0030] 2.2 Effect of quercetin on the expression of thyroid hormone-related genes in MT-exposed gobiocypris rarus As Figure 3The transcription levels of dio1, dio2, tpo, nis, crh in MT group were significantly increased by 170.04%, 119.40%, 173.85%, 68.76%, 61.57% (P<0.05) compared with CON group, while TRa and UGT1a were significantly decreased by 49.39% and 46.44% (P<0.05). TRb had no statistical significance, but had a significant downward trend, reaching 41.37%. The inhibitory effects of 1 ug / L quercetin on the expression of dio2, tpo, crh and nis in MT exposed Gobiocypris rarus were 27.58%, 29.21%, 45.29% and 22.29% (P<0.05), respectively, while the expression of dio1, TRa, TRb and UGT1a was relatively unchanged. The inhibitory effects of 10 ug / L quercetin on the expression of dio1, tpo and crh in MT exposed Gobiocypris rarus were 54.59%, 54.38% and 42.44% (P<0.05), respectively, while the expression of TRa, TRb and UGT1a was promoted by 92.42%, 132.37% and 100.33% (P<0.05), respectively, and the expression of dio2 and nis was relatively unchanged. The inhibitory effects of 100 ug / L quercetin on the expression of dio1, tpo and crh in MT exposed Gobiocypris rarus were 37.25%, 58.38% and 55.33% (P<0.05), respectively, while the expression of TRa, TRb and UGT1a was promoted by 68.00%, 518.90% and 118.75% (P<0.05), respectively, and the expression of dio2 and nis was relatively unchanged. It should be noted that the experimental results in the figure are expressed as mean ± standard error; different lowercase letters represent the significance level (P<0.05).

[0031] 2.3 Effects of quercetin on the expression of serotonin-related genes in MT exposed Gobiocypris rarus As Figure 4The transcription levels of htr1aa, htr1ab, htr1d, tph, tph1 in MT group were significantly higher than those in CON group, with 111.24%, 224.56%, 54.95%, 151.34%, 98.38% (P<0.05). The inhibitory effects of quercetin (1 ug / L) on htr1d and tph1 in MT exposed Gobiocypris rarus were 48.84% and 25.46% (P<0.05), respectively. The expressions of htr1aa, htr1ab and tph were relatively unchanged. The inhibitory effects of quercetin (10 ug / L) on htr1aa, htr1d, tph and tph1 in MT exposed Gobiocypris rarus were 54.51%, 37.35%, 17.23% and 25.97% (P<0.05), respectively, and the expression of htr1ab was relatively unchanged but showed a downward trend. The inhibitory effects of quercetin (100 ug / L) on htr1aa, tph and tph1 in MT exposed Gobiocypris rarus were 33.69%, 16.93% and 45.80% (P<0.05), respectively, and the expression of htr1d was promoted by 120.65% (P<0.05). The expression of htr1ab was relatively unchanged. Note: The test results are expressed as mean ± standard error; different lowercase letters represent significant levels (P<0.05).

[0032] 2.4 Effects of quercetin on T3, T4 and TS levels in MT exposed Gobiocypris rarus To compare with the expression results of genes related to thyroid hormone and serotonin, we quantified the actual levels of serotonin and thyroid hormone in Gobiocypris rarus. As shown in Figure 5 MT group was significantly higher than CON group by 19.77% (P<0.05) and 34.16% (P<0.05). Although there was no significant decrease in T3 content after adding quercetin treatment, there was a clear downward trend. Specifically, at 1, 10 and 100 ug / L, the inhibitory effects were 5.50%, 11.91% and 12.62%, respectively. For T4, at 10 and 100 ug / L, quercetin had a significant inhibitory effect on MT, with inhibitory effects of 19.57% (P<0.05) and 33.45% (P<0.05), respectively. There was no significant difference in the T3 / T4 ratio among the groups. Compared with the CON group, the TS content in the MT group was significantly reduced by 23.35% (P<0.05), and at 1 and 10 ug / L, TS was restored to the CON group level. At 100 ug / L, the TS level was significantly increased by 18.31% (P<0.05). Note: The test results are expressed as mean ± standard error; different lowercase letters represent significant levels (P<0.05).

[0033] 2.6 Changes in brain tissue transcriptome To explore the potential interference effects of quercetin on MT-induced changes in the transcriptional levels of thyroid hormone and serotonin system in Gobiocypris rarus, the brain tissues of CON, MT, and MT+10Q groups were subjected to RNA-seq analysis. The sequencing data of all samples showed high sequencing quality (Q20 > 97%, Q30 > 93%, the number of N bases in single-end read and the percentage of CleanData < 0.03), which could be used for subsequent analysis (Table 2).

[0034]

[0035] The Venn diagram of differentially expressed genes showed the number of unique or common differential genes between each comparison group. As shown in the figure, there were 225 common differential genes between MT and MT+10Q groups compared with the control group; 265 common differential genes between MT+10Q and the control group compared with the MT group; 124 common differential genes between MT and the control group compared with the MT+10Q group; and 35 common differential genes between each two groups of the three groups (Fig. 2). Figure 6 ).

[0036] The volcano plot of differentially expressed genes showed that 461 genes were up-regulated and 346 genes were down-regulated in MT compared with CON. In MT+10Q compared with MT, 138 genes were up-regulated and 462 genes were down-regulated (Fig. 3). Figure 7

[0037] GO is a comprehensive database for describing gene functions, which can be divided into three parts: biological process (BP) and cellular component (CC) molecular function (MF). GO analysis has become an important means of bioinformatics annotation of a gene library. The results showed that the differential genes of MT compared with CON were highly enriched in cell process (189), metabolic process (139), biological regulation process (112), stimulus response (69), cellular anatomical entity (198), binding (243), and catalytic activity (104) in the secondary project. The differential genes of MT compared with MT+10Q were also highly enriched in cell process (137), metabolic process (100), biological regulation process (61), stimulus response (69), cellular anatomical entity (137), binding (181), and catalytic activity (86) (Fig. 4). Figure 8

[0038] ​​KEGG is a comprehensive database of genome-wide functional information. KEGG enrichment analysis showed that the MT group was enriched with 278 pathways compared to the CON group. Among the top 20 significantly enriched pathways, we found significant enrichment in pathways related to neural development and functional maintenance, such as nuclear cytoplasmic transport, apoptosis, and mRNA surveillance pathways (P < 0.05). We also found significant enrichment in pathways related to thyroid hormones, such as the interaction between neuroactive ligands and receptors and the p53 signaling pathway (P < 0.05). Pathways related to serotonin, such as the amyotrophic lateral sclerosis (ALS) pathway and pathogenic Escherichia coli infection, were also significantly enriched. Compared to MT+10Q, MT+10Q was enriched with 275 pathways. Among the top 20 significantly enriched pathways, we found significant enrichment in pathways related to neural development and functional maintenance, such as cell cycle, nuclear cytoplasmic transport, mRNA surveillance pathways, and apoptosis (P < 0.05). Pathways related to serotonin, such as those involved in breast cancer and amyotrophic lateral sclerosis (ALS), were significantly enriched (P < 0.05). Furthermore, we found significant enrichment in pathways related to thyroid hormone synthesis, including N-glycan biosynthesis, mineral absorption, and vitamin digestion and absorption. Figure 9 ).

[0039] III. Interpretation of the Results 3.1 Quercetin alleviates MT-induced thyroid hormone disorders in rare gudgeon. Thyroid follicular tissue with a normal cellular structure facilitates iodine uptake and thyroid hormone biosynthesis, while pathological changes in follicular tissue can disrupt thyroid hormone homeostasis. Previous studies have shown that T4 and T3 are synthesized and secreted by thyroid follicles, and thyroid hormone secretion disorders may occur when thyroid follicular cells proliferate and colloid is lost. In this study, MT induced significant proliferation and hypertrophy of thyroid follicular epithelial cells in rare gudgeon, even colloid loss, and abnormally elevated T3 and T4 levels. A previous study indicated that pathological damage to the thyroid gland may lead to the release of large amounts of T4, which is converted into active T3. This suggests that MT can cause pathological damage to thyroid follicles and disrupt thyroid hormone homeostasis. In the quercetin treatment group, a concentration of 10 U / L of quercetin effectively improved thyroid follicular hyperplasia and hypertrophy, and T3 and T4 levels returned to normal. This suggests that appropriate quercetin can alleviate thyroid damage caused by MT, thereby maintaining thyroid hormone homeostasis.

[0040] Changes in the expression of thyroid-related genes caused by environmental pollutants are also considered as one of the markers of thyroid dysfunction. CRH stimulates thyroid hormone secretion in teleosts. NIS transports iodine from the blood into the colloid of thyroid follicles, and TPO binds iodine to tyrosine residues on the thyroglobulin (TG) molecule to produce T4. UGT1a gene functions to inactivate T4 metabolically and excrete it into the bile. In this study, MT caused an increase in the transcriptional levels of CRH, NIS, and TPO, and a decrease in the transcriptional level of UGT1a, which might be the reason for the increase in T4 content in the MT group. The metabolism of T4 to T3 requires Dio1 and Dio2, in which Dio1 is involved in the degradation of thyroid hormones, and Dio2 converts T4 to T3, which is more biologically active. MT caused an increase in the transcriptional levels of DIO1 and DIO2, which means that although T4 increased, more T3 was also produced, which also explains why there was no significant difference in the T3 / T4 ratio in the MT group. In addition, quercetin treatment reversed the MT exposure-induced changes in the transcriptional levels of CRH, NIS, TPO, UGT1a, DIO1, and DIO2 in G. a. rarebook, indicating that quercetin can protect G. a. rarebook from MT-induced transcriptional abnormalities of thyroid-related genes. And from the effect of relief, the treatment effect of 10 ug / L concentration of quercetin is the best.

[0041] Thyroid hormone signaling is the result of the interaction of nuclear thyroid hormone receptors (TRs) with specific target gene promoters, a process that can either enhance or inhibit transcription. Environmental endocrine disruptors interfere with the homeostasis of thyroid hormones by binding to thyroid receptors and altering their normal signaling process. Our results showed that the mRNA expression of TRa and TRb decreased in G. a. rarebook after 28 days of MT exposure, and the same experimental results also occurred in zebrafish. Perfluorinated sulfonate has been shown to bind to thyroid hormone receptors, and its exposure experiment on G. a. rarebook also showed that its TRs transcription level was inhibited and the T3 level was increased. Therefore, we speculate that MT, as an endocrine disruptor, can bind to thyroid hormone receptors and interfere with normal thyroid hormone signaling, such as changes in the composition of the transcription complex leading to a decrease in its transcription level. The increase in T3 may be the result of decreased receptor expression. Quercetin can effectively reverse the changes in TRa and TRb caused by MT, thereby regulating the efficiency of target tissues in utilizing thyroid hormones.

[0042] 3.2 Quercetin alleviates MT-induced serotonin disorder in G. a. rarebook 5-HT neurotransmitter system is highly conserved in vertebrates, which plays a role in animal behavior, gonadal development and reproduction and other physiological activities by initiating a cascade reaction after binding with serotonin receptors. A previous study on female rainbow trout showed that MT can cause serotonin to decrease. This is consistent with the results of this study. In addition, the expression of serotonin receptors 5ht1aa, 5ht1ab and 5ht1d in MT-treated Gobiocypris rarus increased, suggesting that MT exposure led to overexpression of serotonin receptors, which in turn reduced the content of serotonin. Studies on human patients with depression have shown that depression is associated with an increase in 5-HT 1A receptor levels. Changes in the expression of 5ht1aa and 5-htr1ab genes may have an important impact on the neurobehavior of Gobiocypris rarus. Tph is the rate-limiting enzyme in 5-HT biosynthesis. Our data further showed that the expression of tph and tph1 was up-regulated after MT treatment, which may be a feedback regulation of the body after the decrease of serotonin levels. In the quercetin treatment group with a concentration gradient, the 1 and 10 ug / L quercetin groups reversed the transcription levels of htr1d, htr1aa and tph1 in MT-exposed Gobiocypris rarus, bringing the serotonin to normal levels. However, in the 100 ug / L quercetin group, the transcription level of htr1d and the level of serotonin were abnormally high. This suggests that adding an appropriate concentration of quercetin can alleviate the serotonin disorder caused by MT.

[0043] 3.3 Effect of quercetin on the brain transcriptome of MT-exposed Gobiocypris rarus To further explore the effect of quercetin on alleviating the interference of MT on serotonin and thyroid hormone, this paper analyzed the changes in the transcription level of Gobiocypris rarus after exposure in each group by RNA-seq sequencing technology, and screened the possible toxic pathways of MT on serotonin and thyroid hormone and the alleviating pathways of quercetin.

[0044] As mentioned earlier, thyroid hormone and serotonin play a crucial role in fish neurobehavior. Pathways that affect the normal development and functional maintenance of neurons, such as nuclear-cytoplasmic transport, apoptosis, mRNA surveillance pathway, were found in the MT and MT+10Q groups, suggesting that the alleviation of thyroid hormone and serotonin disorder caused by MT by quercetin is related to brain nerve damage.

[0045] For thyroid hormones, we found that the MT and MT+10Q aggregated thyroid hormone-related pathways were different. MT found the neuroactive ligand-receptor interaction and p53 signaling pathway, while MT+10Q found the N-glycan biosynthesis, mineral absorption, and vitamin digestion and absorption pathways. Other studies have also found that these four pathways are related to thyroid hormones. The p53 signaling pathway is involved in multiple thyroid pathways, including thyroid hormone signaling and thyroid cancer. The p53 transcription factor is a key molecule for identifying thyroid cancer and can interfere with TRs and inhibit the transactivation activity of TRβ. Exposure to decabromodiphenyl ether in rats and zebrafish found that significant changes in the neuroactive ligand-receptor interaction signaling pathway contributed to the occurrence of thyroid disruption. N-glycan biosynthesis is closely related to thyroid hormone synthesis. Both the pituitary-secreted thyroid-stimulating hormone (TSH) and the thyroid hormone receptors on the surface of thyroid cells contain n-glycans. Changes in these n-glycans can lead to abnormal thyroid activity and changes in hormone metabolic clearance, resulting in thyroid disease. Adequate intake of trace elements and vitamins helps maintain normal thyroid metabolism.

[0046] Serotonin (5-hydroxytryptamine, 5-HT) is an important endogenous substance found to be involved in the development of ALS. We found that both MT and MT+10Q aggregated the ALS pathway, which may be another piece of evidence that quercetin repairs the nerve damage to the brain caused by MT.

[0047] These findings provide important molecular basis for the mechanism by which MT affects thyroid hormone, serotonin homeostasis, and quercetin alleviation.

[0048] In summary, long-term exposure to MT can cause brain nerve damage in Gobiocypris rarus, leading to thyroid hormone and serotonin secretion disorders. Quercetin, as a natural substance with multiple biological activities, can alleviate the toxic effects of MT by repairing nerve damage, restoring thyroid tissue structure, balancing hormone levels, and other pathways. These research results provide a new perspective for quercetin to alleviate the effects of MT on thyroid hormone and serotonin secretion disorders in Gobiocypris rarus. Future research can explore the effects of quercetin on other aquatic organisms under different EDCs and its mechanisms of action.

[0049] Example Two: The present application finds the application of quercetin in aquaculture. Quercetin can be used as a drug to prevent and treat thyroid hormone and / or serotonin disorders in aquatic animals. Quercetin can also be added to feed, which can help regulate thyroid hormone and serotonin levels to normal levels while providing daily nutritional supplements. This method of diet therapy is safe and healthy.

[0050] The aquatic animal is rare gobiocypris rarus, the mass concentration of quercetin is 1 μg / L, the medicine further comprises a medicine lipid carrier acceptable to quercetin, the lipid carrier is composed of apolipoprotein A-1 and lecithin, the mass ratio of apolipoprotein A-1 and lecithin is 1:2, and the mass ratio of the liposome and quercetin is 30:20.

[0051] The preparation method of the quercetin wrapped lipid carrier adopts microfluidic preparation. (1) Preparation of inorganic phase: apolipoprotein A-1 single analog peptide is added into inorganic reagent to dissolve, to obtain solution A; (2) Preparation of organic phase solution: quercetin and lecithin are added into organic reagent to dissolve, to obtain solution B; (3) The solution A and the solution B are mixed by using microfluidic to prepare the medicine.

[0052] In the step (1), the apolipoprotein A-1 single analog peptide is dissolved in PBS, and the mass:volume of the apolipoprotein A-1 single analog peptide and PBS is 1 mg:10 ml; In the step (2), the organic reagent is ethanol, and the mass:mass:volume of the quercetin, lecithin and ethanol is 0.1 mg:2 mg:5 ml.

[0053] In the step (3), the microfluidic method is that the mixture of the quercetin wrapped lipid carrier is synthesized according to the flow rate of 1 mL / min of the organic phase and 5 mL / min of the inorganic phase.

[0054] In the application, quercetin is used as the application of preparing a medicine for preventing and treating thyroid hormone disorder and / or serotonin disorder in aquatic animals, and quercetin can also be added into feed, so that the thyroid hormone and serotonin can be restored to normal level while nutrients are supplemented daily, and the method of diet therapy is safe and healthy.

[0055] The aquatic animal is rare gobiocypris rarus, the mass concentration of quercetin is 1 μg / L, the medicine further comprises a medicine lipid carrier acceptable to quercetin, the lipid carrier is composed of apolipoprotein A-1 and lecithin, the mass ratio of apolipoprotein A-1 and lecithin is 1:2, and the mass ratio of the liposome and quercetin is 30:20.

[0056] The preparation method of the quercetin wrapped lipid carrier adopts microfluidic preparation. The method comprises the following steps: (1) Preparation of inorganic phase: apolipoprotein A-1 single analog peptide is added into inorganic reagent to dissolve, to obtain solution A; (2) Preparation of organic phase solution: quercetin and lecithin are added into organic reagent to dissolve, to obtain solution B; (3) using microfluidic, solution A, solution B mixed to prepare the drug.

[0057] The apolipoprotein A-1 single simulation peptide in step (1) is dissolved with PBS, and the mass: volume of the apolipoprotein A-1 single simulation peptide and PBS is 5 mg: 10 ml; The organic reagent in step (2) is ethanol, and the mass: mass: volume of quercetin, lecithin and ethanol is 0.5 mg: 8 mg: 10 ml.

[0058] The microfluidic method in step (3) is to synthesize a mixture of quercetin wrapped lipid carrier according to the flow rate of 5 mL / min of organic phase and 10 mL / min of inorganic phase.

[0059] Example four: the application of quercetin in aquaculture is found in the application, quercetin is used as an application for preparing a drug for preventing and treating thyroid hormone disorder and / or serotonin disorder of aquatic animals, and quercetin can also be added to feed, so that the nutrients can be supplemented daily, and the thyroid hormone and serotonin can also be adjusted to normal level. This method of diet therapy is safe and healthy.

[0060] The aquatic animal is rare Procyprinus, the mass concentration of quercetin is 50 μg / L, the drug further comprises a drug lipid carrier acceptable to quercetin, the lipid carrier is composed of apolipoprotein A-1 and lecithin, the mass ratio of apolipoprotein A-1 to lecithin is 3:4, and the mass ratio of the lipid carrier to quercetin is 40:30.

[0061] The preparation method of the quercetin wrapped lipid carrier adopts microfluidic preparation. The method comprises the following steps: (1) preparing an inorganic phase: dissolving the apolipoprotein A-1 single simulation peptide in an inorganic reagent to obtain solution A; (2) preparing an organic phase solution: dissolving quercetin and lecithin in an organic reagent to obtain solution B; (3) using microfluidic, solution A, solution B mixed to prepare the drug.

[0062] The apolipoprotein A-1 single simulation peptide in step (1) is dissolved with PBS, and the mass: volume of the apolipoprotein A-1 single simulation peptide and PBS is 3 mg: 10 ml; The organic reagent in step (2) is ethanol, and the mass: mass: volume of quercetin, lecithin and ethanol is 0.3 mg: 4 mg: 7 ml.

[0063] The microfluidic method in step (3) is to synthesize a mixture of quercetin wrapped lipid carrier according to the flow rate of 3 mL / min of organic phase and 7 mL / min of inorganic phase.

[0064] Embodiment five: the application of quercetin in aquaculture is discovered in the application, quercetin is used as an application of preparing a medicine for preventing and treating thyroid hormone disorder and / or serotonin disorder of aquatic animals, and quercetin can also be added to feed, so that the thyroid hormone and serotonin can be restored to normal levels while daily nutrition is supplemented, and the method of diet therapy is safe and healthy.

[0065] The aquatic animal is rare gobiocypris rarus, the mass concentration of quercetin is 25 μg / L, the medicine further comprises a drug lipid carrier acceptable to quercetin, the lipid carrier is composed of apolipoprotein A-1 and lecithin, the mass ratio of apolipoprotein A-1 to lecithin is 4:5, and the mass ratio of the lipid carrier to quercetin is 45:25.

[0066] The preparation method of the quercetin wrapped lipid carrier adopts microfluidic preparation, and the method comprises the following steps: (1) preparing an inorganic phase: apolipoprotein A-1 single analog peptide is added into an inorganic reagent to obtain solution A; (2) preparing an organic phase solution: quercetin and lecithin are added into an organic reagent to obtain solution B; (3) mixing solution A and solution B by microfluidic to obtain the medicine.

[0067] In the step (1), the apolipoprotein A-1 single analog peptide is dissolved in PBS, and the mass:volume of the apolipoprotein A-1 single analog peptide to PBS is 4 mg:10 ml; In the step (2), the organic reagent is ethanol, and the mass:mass:volume of quercetin, lecithin and ethanol is 0.4 mg:7 mg:9 ml.

[0068] In the step (3), the microfluidic method is that the mixture of quercetin wrapped lipid carrier is synthesized according to the flow rate of 4 mL / min of the organic phase and 9 mL / min of the inorganic phase.

[0069] The following part illustrates the creative labor of the applicant when the medicine wrapped by the quercetin lipid carrier is obtained by listing some experimental examples: Experimental method: common lipid carrier materials such as dipalmitoyl choline, phosphatidylcholine and phosphatidylglycerol are set as comparative examples, the rare gobiocypris rarus is exposed to a water environment of 50 ng / L MT for 7 days, the thyroid hormone level TSH, T3, T4 and the serotonin level of the gobiocypris rarus are detected, then the quercetin liposome of comparative examples 1-3 is intervened according to the application embodiments two to five, the values of the above hormones after intervention for 0 days, 7 days and 14 days are detected, and the following results are obtained:

[0070] It can be seen from the above results that the liposome carrier is used in the application, and the best effect can be achieved. It should be noted that the average results of 3 parallel repeated experiments are obtained for each group, and the existing technology is used in the experimental process. In order to save space, a simplified method is used for description. The method for preparing liposomes in Comparative Examples 1-3 uses the existing technology, for example, quercetin is dissolved in the liposome solution, and reverse evaporation is used for encapsulation, and then the liposome-encapsulated quercetin material is obtained by purification, sterilization and other methods.

[0071] The above is the preferred embodiment of the application. It should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the application. These improvements and refinements should also be considered within the scope of protection of the application.

Claims

1. Use of quercetin in aquaculture, characterized in that, The application of quercetin in the preparation of medicines and / or feeds for preventing and treating thyroid hormone disorder and / or serotonin disorder of aquatic animals.

2. Use according to claim 1, characterized in that, The aquatic animal is rare gobiocypris rarus.

3. Use according to claim 1, characterized in that, The mass concentration of quercetin is 1-100 μg / L.

4. Use according to claim 1, characterized in that, When quercetin is used in the preparation of medicines for preventing and treating thyroid hormone disorder and / or serotonin disorder of aquatic animals, the medicines further comprise a drug lipid carrier acceptable to quercetin.

5. Use according to claim 4, characterized in that, The lipid carrier is composed of apolipoprotein A-1 and lecithin, and the mass ratio of apolipoprotein A-1 to lecithin is 1-5:2-6, and the mass ratio of the lipid carrier to quercetin is 30-50:20-50.

6. Use according to claim 5, characterized in that, The preparation method of the quercetin lipid carrier adopts microfluidic technology.

7. Use according to claim 6, characterized in that, The method comprises the following steps: (1) preparing an inorganic phase: adding apolipoprotein A-1 single analog peptide into an inorganic reagent to obtain solution A; (2) preparing an organic phase solution: adding quercetin and lecithin into an organic reagent to obtain solution B; (3) mixing solution A and solution B by microfluidic technology to obtain the medicine.

8. Use according to claim 7, characterized in that, In step (1), the apolipoprotein A-1 single analog peptide is dissolved in PBS, and the mass:volume of the apolipoprotein A-1 single analog peptide and PBS is 1-5 mg:10 ml; In step (2), the organic reagent is ethanol, and the mass:volume of quercetin, lecithin and ethanol is 0.1-0.5 mg:2-8 mg:5-10 ml; In step (3), the microfluidic method is to synthesize the quercetin lipid carrier mixture according to the flow rate of the organic phase 1 mL / min-5 mL / min and the flow rate of the inorganic phase 5 mL / min-10 mL / min. ​

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