CN110447787B - Application of galanthamine in preventing and treating fish enteritis caused by bean pulp feed - Google Patents

Abstract

The invention discloses an application of galanthamine in prevention and treatment of bean pulp induced fish enteritis, which conjectures that galanthamine is a potential drug for prevention and treatment of bean pulp induced enteritis according to a neural regulation theory of intestinal mucosa immunity and two methods of disease modeling and biological information analysis; experiments prove that the galanthamine can activate the expression of the anti-inflammatory factors in the intestinal tract of the cultured fish, inhibit intestinal inflammation, maintain the close connection of intestinal epithelial barriers and inhibit the apoptosis of the intestinal epithelium no matter the galanthamine is added into the feed or injected into the back muscle, so that the enteritis caused by the bean pulp feed is relieved, the fish can recover the growth, and the galanthamine can be used as a novel feed additive or injection needle with the anti-inflammatory effect.

Description

Application of galanthamine in preventing and treating fish enteritis caused by bean pulp feed

Technical Field

The invention belongs to the field of veterinary science, and particularly relates to application of galanthamine in prevention and treatment of fish enteritis induced by soybean meal.

Background

Aquatic animal enteritis is one of the major diseases causing economic loss of aquaculture. Aquatic animals, especially fishes and shrimps, need to be added with animal protein such as fish meal and the like in the feed, however, the annual shortage of fish meal resources becomes a key factor limiting the sustainable development of the aquaculture industry in China, so that the use of a plant protein source to replace the fish meal and improve the utilization rate of the fish meal is a necessary choice for the development of the aquaculture industry. However, when the ratio of the vegetable protein source such as soybean meal and cottonseed meal to the fish meal is too high, fish intestinal inflammation, liver pathological changes and growth reduction are generally induced, so that the application of the vegetable protein source in aquatic animal feed is limited. Research shows that the maximum substitution amount of the soybean meal in the feed for herbivorous fishes, grass carps and omnivorous fishes is 60 percent, and the maximum substitution amount of the soybean meal in the feed for omnivorous fishes, carps and clarias fuscus is 45 percent. The substitution amount of the soybean meal in the carnivorous cobia reaches 10 percent to the maximum extent, and the soybean meal has no influence on health, and has obvious influence on growth, weight gain rate, feed coefficient, survival rate and fullness when the substitution proportion is 20 percent to 30 percent. The current research on the response and immunoregulation of the fish intestinal mucosal immune system to plant protein sources has mainly focused on soybean meal-induced enteritis (SBMIE) and its growth inhibition. Fish for which symptoms of SBMIE have been reported include salmon and trout, such as Atlantic salmon and rainbow trout; carps, such as zebrafish and carp; catfish, such as yellow catfish. In the SBMIE process, the research on proinflammatory factors and inflammatory response thereof is common, the immune mechanism for relieving/inhibiting inflammation and the corresponding pharmacological research are still in the initial stage, and the effect is unstable.

At present, antibiotics and traditional Chinese medicine preparations with broad-spectrum therapeutic effects are mostly used in production, and medicines for treating the targeted enteritis are very lacking. A large amount of antibiotic drugs are used for a long time, so that drug-resistant strains are easily generated, the enteritis of fishes in local areas is frequently outbreak, and the phenomena of increased dosage, long-term treatment and the like occur. The increase of the dosage and the drug residue also cause potential harm to human health of aquatic animals. Therefore, antibiotics are urgently needed to be replaced, clues are searched from the intestinal mucosa immunity mechanism of the fish body, and the aim of treating enteritis of fishes without public hazard is achieved.

The enteric nervous system (enteric neurous system) is present in the intestinal mucosa of fish and interacts with the immune system to form an independent integrated system that regulates and controls the function of the digestive tract. Lymphocytes in the intestinal submucosa, such as T, B lymphocytes, also including innate lymphocytes, are in intimate contact with nerve fibers, and there are local loops of immune and neural interactions. The interaction between nerve cells and immune cells depends on the contact of cells and soluble molecules, such as signal transmission of cytokines, chemokines, neuropeptides, neurotrophic factors and the like, and the immune cells can express receptors of neurotransmitters, so that the neurotransmitters can be sensed and immune responses can be regulated.

The cholinergic anti-inflammatory pathway (CAIP) plays an important role in the neuroimmune loop of the intestinal tract, and the anti-inflammatory response time is very short, so that the cholinergic anti-inflammatory pathway can rapidly and directly regulate the systemic inflammatory response. After a vagus nerve or immune cells with choline acetyltransferase (ChAT) are activated, acetylcholine can be released, immune cells such as T/B lymphocytes, macrophages, granulocytes and dendritic cells are stimulated through an acetylcholine receptor (nAChR-alpha7), and release of proinflammatory factors is inhibited, so that the immunosuppressive effect is achieved; while the activation of nAChR-alpha7 affects immune cells, namely NF-. kappa.B and Jak/STAT pathways, mainly through 2 signal pathways. Recent studies have found that regulatory T cells also express nAChR-alpha7, and that this receptor, when activated, enhances immunosuppressive functions. Activation of nAChR-alpha7 can inhibit T cell proliferation and Th1 and Th17 cytokine production, promote Th1 to Th2 shift, and thus achieve immunoregulation effect. For B lymphocytes, choline receptors are mainly involved in promoting the expression of B cell costimulatory molecules, promoting the conversion of pre-B cells into mature B cells, and the proliferation of B cells.

Galantamine, a common potent and reversible acetylcholinesterase inhibitor, can enhance acetylcholine pathway by inhibiting acetylcholine degradation, and thus can be used for treating alzheimer's disease (approved by FDA) and also for treating severe myasthenia. Regarding its anti-inflammatory action, only certain therapeutic effects on the concentration blood disease are seen at present. Thus, we speculate that galantamine may have a potentiating effect on the cholinergic anti-inflammatory effect in intestinal inflammation, which has potential application for the treatment of inflammatory bowel disease in aquatic animals. Galantamine has a clearance half-life in vivo of 7-8 hours, is metabolized by the liver, and is cleared by excreta, so that it can be prevented from remaining in aquatic animal products as long as use is stopped. Common application forms of galanthamine are hydrobromide, N-terminal alkyl modification, and some resin-added slow-release solution forms.

The invention firstly uses the cholinergic anti-inflammatory pathway theory for the treatment of cultured fishes, and finds that the acetylcholinesterase inhibitor galanthamine can relieve enteritis caused by bean pulp feed. Specifically, the galanthamine has the effects of reducing the expression level of inflammatory factors, recovering the expression level of intestinal mucosa tight junction protein and inhibiting the expression of apoptosis genes of intestinal epithelium, so that the effects of resisting inflammation and repairing the intestinal mucosa barrier are achieved, and the growth is not influenced. Based on the discovery and application experience of model organisms of zebra fish and cultured fish grass carp, the galanthamine can be used for preventing and treating enteritis of cultured fish caused by bean pulp feed, and can replace the currently common antibiotic enteritis drugs, reduce environmental pollution and drug residue, and has wide application prospect in the field of aquatic products.

Disclosure of Invention

The invention aims to provide application of galanthamine in preventing and treating enteritis of cultured fishes caused by bean pulp feed, the galanthamine can be used for preventing or treating enteritis of fishes induced by bean pulp in aspects of reducing proinflammatory factors, improving expression of anti-inflammatory factors, improving intestinal barrier function and the like, and the application method of the galanthamine comprises the step of using the galanthamine as a feed additive and an injection.

In order to achieve the purpose, the invention adopts the following technical measures:

the application of galanthamine as a feed additive in relieving enteritis of cultured fishes caused by soybean meal feed comprises the following steps:

0.5-1.0 ppm of galanthamine is added into feed with soybean meal replacing 50% of protein source, after the feed is continuously fed with omnivorous fishes, namely zebra fishes for 3 weeks (once every morning and evening), MS-222 is used for anesthesia, intestinal tract samples are dissected and taken, and the prevention and treatment effects are tested through qPCR, Western blot, H.E. staining and immunohistochemistry. The result shows that the galanthamine with the addition concentration of 0.5-1ppm in the feed has the effect of relieving enteritis caused by soybean meal in the aspects of morphological level, growth level, gene expression (including immunity, tight connection, apoptosis) and the like, and can promote the recovery of feed intake of fish bodies.

The application of galanthamine in preparing the medicine for preventing or treating enteritis of cultured fishes caused by bean pulp feed comprises the following steps:

injecting galantamine (0.05-0.1 mg/kg of fish body weight) into the back muscle of a sick grass carp (the soybean meal replaces 70% of feed of a protein source for 7 weeks), injecting for 2 times every day for 2 days, and taking an intestinal tissue sample on the 4 th day to perform fluorescence quantitative qPCR analysis to detect the expression condition of the immune gene related to the intestinal inflammation. The results show that galantamine injection can significantly reduce the expression level of inflammation-associated immune genes including proinflammatory factors IL-1b and IL17, a T cell surface marker CD4 and a regulatory lymphocyte transcription factor Foxp3 in SBMIE grass carp intestinal tissues compared with diseased fish injected with PBS. Therefore, in the fish body suffering from enteritis caused by bean pulp, galantamine with a certain injection dose can play a role in inhibiting enteritis by inhibiting the expression of inflammatory factors, and further relieve the disease condition.

Through the evolution analysis of cholinesterase protein and acetylcholine receptor nAChR-alpha7 protein of common cultured fish, the cholinesterase which is the substrate of galanthamine widely exists in the fish, and is very conservative compared with the sequence of mammals; on the other hand, common cultured fishes have acetylcholine receptors which are necessary for choline-activated anti-inflammatory pathways. Therefore, the galanthamine also has a protective effect on enteritis caused by bean pulp of cultured fishes with acetylcholine receptors. The fishes related to the invention specifically comprise fishes of the family Cyprinidae, such as grass carp, herring, silver carp, bighead carp, crucian carp, megalobrama amblycephala and the like, salmon and trout, such as Atlantic salmon, rainbow trout and the like, common farmed fishes such as tilapia, channel catfish and the like, and are particularly suitable for herbivorous and omnivorous farmed fishes.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the invention provides the application of galanthamine in preventing and treating enteritis of cultured fishes caused by bean pulp feed for the first time, the adopted galanthamine is used as a medicine for realizing anti-inflammatory action by utilizing a cholinergic anti-inflammatory pathway of a fish body, when the galanthamine is used as a fish feed additive, the problems of residue of antibiotic medicines and environmental safety are avoided, no toxic or side effect is caused, the galanthamine meets the green fishery medicine standard, can be widely applied to feeds containing plant protein sources such as bean pulp and the like, and is used for improving the intestinal mucosa immunity of aquatic animals, preventing enteritis caused by the feeds, further improving the growth efficiency, and simultaneously reducing the cost compared with adding fish meal. Whether the galanthamine is added into fish feed or is injected intramuscularly, the galanthamine can activate the expression of anti-inflammatory factors in intestinal tracts of cultured fishes, inhibit intestinal inflammation, maintain the close connection of intestinal epithelial barriers and inhibit the apoptosis of the intestinal epithelia, so the galanthamine can be used as a novel feed additive with an anti-inflammatory effect or an injection needle.

Drawings

FIG. 1 is a graph showing the results of polyclonal antibody specific detection of IL17, IL10, and TGF-beta. The respective antigens of IL17, IL10 and TGF-B expressed by pronucleus are effectively purified by affinity chromatography column (A), and the polyclonal antibodies of IL17, IL10 and TGF-B are obtained by immunizing rabbit with purified antigen, and can specifically recognize the corresponding natural protein (B) in fish body.

FIG. 2 shows that acetylcholine anti-inflammatory pathway is activated in intestinal transcriptome after various stages of enteritis induced by grass carp soybean meal. In the KEGG pathway of neurotransmitter interaction with receptors (neuroactive ligand-receptor interaction), the first neurotransmitter in the first column on the left is Acetylcholine, and the grey frame is the gene for detecting the transcript.

FIG. 3 shows the similar drug expression profile of galantamine in the CMap database.

FIG. 4 shows the expression profile of galantamine for the combination of NGF + HotNet + CMap.

Fig. 5 shows the recovery of tissue morphology of the hindgut of zebra fish bean pulp feed after the addition of galanthamine with different concentrations. FM: fish meal feed group (negative control); SBM: soybean meal feed group (positive control); SBM +0.5ppm Gal: a soybean meal feed group to which 0.5ppm of galanthamine is added; SBM +1ppm Gal: a soybean meal feed group added with 1ppm of galanthamine.

Fig. 6 is the recovery situation of the growth index of zebra fish bean pulp feed added with galanthamine of different concentrations. IBM: initial body weight, FBW: final body weight, WG: total weight gain, FI: feed intake, FE: feed efficiency, SGR: a specific growth rate. Soybean meal served as a positive control for enteritis, while fish meal served as a negative control. The plum blossom or well symbol indicates a significant difference p <0.05, the former representing up-regulation compared to the fish meal feed group and the latter representing down-regulation compared to the soybean meal feed group.

FIG. 7 shows the expression of inflammation-related immune genes in the hindgut tissue after the zebra fish bean pulp feed is added with galantamine at different concentrations. A: qPCR, the vertical axis is relative expression quantity change multiple, and the horizontal axis is different groups; b: western blot, wherein the expression condition of enteritis-related immune protein caused by bean pulp feed is shown in a box; c: immunohistochemistry was performed to examine the localization of CD4 protein in the posterior intestinal mucosal tissue. Soybean meal served as a positive control for enteritis, while fish meal served as a negative control. The quincunx or the well indicates a significant difference p <0.05, the former representing a significant up-regulation compared to the fish meal feed group and the latter representing a significant down-regulation compared to the soybean meal feed group.

FIG. 8 shows the expression of genes related to intestinal epithelial tight junction (A) and apoptosis (B) after different concentrations of galantamine are added to the zebra fish bean pulp feed. Soybean meal served as a positive control for enteritis, while fish meal served as a negative control. The quincunx or well number indicates a significant difference p <0.05, the former representing upregulation compared to the FM group and the latter representing downregulation compared to the SBM group.

FIG. 9 shows the immune gene expression changes after injecting galantamine into diseased grass carp (70% feed replacing protein source for 7 weeks). FM: a fish meal feed group; 70% of soybean meal: a soybean meal feed group which completely replaces fish meal. Also lower case letters indicate no significant difference p >0.05, different lower case letters indicate significant difference p < 0.05.

FIG. 10 is an NJ tree evolutionary analysis of the cholinesterase protein of fish. The zebrafish cholinesterase gene (ENSDARG00000031796.8) and the grass carp cholinesterase gene (CI _ GC _7222) are obtained by translation using the coding region sequences thereof. The sequence numbers of the cholinesterase protein downloaded by NCBI are respectively: human (NP _ 000046.1); mouse (NP _ 033868.3); silversmith (XP _ 023648146.1); carp (KTG 31068.1); medaka (NP _ 001188442.1); tilapia (XP _ 003441667.2); finless eel (XP _ 020456087.1); large yellow croaker (KKF 12911.1). And the sequence number of zebrafish acetylcholinesterase (ChAT) selected as the foreign group is NP-001124191.1.

FIG. 11 is an NJ tree evolution analysis of the acetylcholine receptor nAChR-alpha7 protein of common farmed fish. The protein nAChR-alpha7 except the grass carp comes from grass carp genome database of aquatic organism research institute of Chinese academy of sciences, and the gene numbers are CI _ GC _10747 and CI _ GC _ 18333; other fish are derived from NCBI and have the sequence numbers: zebrafish nAChR-alpha7 protein (AAO91913.1), zebrafish nAChR-alpha7-like protein (XP _005174278.4), carp nAChR-alpha7 homologous protein (KTF81060.1), herring nAChR-alpha7 protein (XP _012674679.1), barbel nAChR-alpha7 protein (XP _016140050.1), Atlantic salmon nAChR-alpha7 protein (XP _013979444.1), rainbow trout nAChR-alpha7 protein (XP _021421301.1), arctic salmon nAChR-alpha7 protein (XP _023840673.1), catfish nAChR-alpha7 protein (XP _017332872.1), finless nAChR-alpha7 protein (XP _020461482.1), large yellow croaker nAChR-7 protein (XP _019113632.1), and tilapia nAChR-alpha7 protein (XP _ 003450802.1). The human foreign group has the protein sequence number of AAA83561.1 of nAChR-alpha 7.

Detailed Description

The invention is further described with reference to the following drawings and specific examples, which should not be construed as limiting the invention. All molecular biological manipulations referred to in the examples are, unless otherwise specified, conventional procedures well known to those skilled in the art.

The test materials, reagents and instrumentation referred to in the examples are as follows:

polyclonal antibodies of cytokines IL17, IL10 and TGF-beta of grass carp are prepared by the laboratory, and the method comprises the following steps:

(1) selecting an antigen fragment: removing all the rest protein sequences of the signal peptide, wherein the antigen fragment of the grass carp IL17(NCBI access: KC978892.1) is from 28 th amino acid to C terminal, the antigen selection of the grass carp IL10(NCBI access: JQ768312.1) is from 25 th amino acid to C terminal, and the antigen fragment of grass carp TGF-beta (NCBI access: EU099588.1) is from 266 th amino acid to 377 th amino acid;

(2) selecting an expression vector: IL17 and TGF-beta adopt GST-tag label expression vector PGEX-4T-1, IL10 adopts his-tag label expression vector PET-23 b;

(3) obtaining a recombinant protein: prokaryotic expression is carried out in escherichia coli DE3, recombinant proteins of IL17 (soluble), IL10 (inclusion body renaturation is soluble) and TGF-beta (soluble) are obtained, and the sizes of the recombinant proteins are respectively 17kDa, 19.7kDa and 15.3kDa (figure 1A);

(4) immunizing animals to obtain antibodies: injecting recombinant protein solution (600 μ g/ml) into rabbit, immunizing 3 times (1 ml each time, 2 weeks apart), and injecting for the first immunization: popliteal lymph node and plantar region of the foot subcutaneously, second and third times: injecting subcutaneously at multiple points on the back, and taking serum 1 week after the third immunization to obtain polyclonal antibody;

(5) the antibody specificity verification result shows that: the fact that IL17 antibody recognizes a single band with the size of about 22kDa, IL10 antibody recognizes a single band with the size of about 20kDa and TGF-beta antibody recognizes a single band with the size of about 15kDa in peripheral blood leukocytes isolated from fish bodies is equivalent to the predicted size proves that the antibodies prepared by the method can specifically recognize the corresponding proteins in fish bodies.

The zebrafish Foxp3 and CD4 used in the present invention are products of GeneTex (numbered GTX16590 and GTX16589, respectively), h.e. dye liquors (petunia), kits for extracting RNA (TIANGEN biochemicals, beijing) limited), primary antibodies (zebrafish CD4 rabbit polyclonal antibody from GeneTex, zebrafish Foxp3 rabbit polyclonal antibody from Creative Diagnostics, Trizol (invitrogen),

the kit (Thermo Fisher Scientific), protein extract (Protease Inhibitor Cocktails), WB secondary antibody and fluorescent secondary antibody were purchased from Sigma-Aldrich, all qPCR primers were provided synthetically by wuhan technologies services, all chemicals were from national reagents.

Example 1 prediction of efficacy of galanthamine as a functional ingredient for relieving enteritis

KEGG pathway analysis of grass carp transcriptome suggests the existence of fish cholinergic anti-inflammatory pathways: the inventors' existing studies indicate that 40% of the soybean meal has a self-restorable ability to replace intestinal inflammation in fish meal group grass carp starting from week 5 to week 7. Transcriptomes were measured at various stages of the disease course including 0 day, 3 weeks, 5 weeks, and 7 weeks, and after intestinal tissue was taken, wherein analysis results in KEGG pathway showed that Acetylcholine (acetylcholinene) pathway was activated during occurrence and resolution of soybean meal-induced enteritis (fig. 2), suggesting that a cholinergic anti-inflammatory pathway may exist in fish.

Then, we used CMap database to predict the efficacy of the cholinesterase inhibitor galanthamine: galantamine as a cholinesterase inhibitor may promote the function of the cholinergic anti-inflammatory pathway, and in order to further infer its possible role in enteritis, the inventors used the CMap database for efficacy prediction. The Connectivity map (CMap) (https:// ports. branched. organization/CMap /) is a gene expression profile database, and a biological application database of the small molecule drugs, the gene expression and the diseases which are correlated with each other is established by utilizing the gene expression difference of the small molecule drugs after the human cells are treated. In the field of drug development, the inventor can quickly compare the data of gene expression profiles to obtain drugs with high association with diseases, and can summarize the mechanism direction of possible action of drug molecules. The galanthamine was retrieved in the CMap library and the MCF7 cell line was selected, and the up/down threshold was set at. + -. 0.67(2-fold) to give the drug the most similar to the galanthamine expression profile. As shown in fig. 3, the top four drugs with the highest P-value and the top mean in CMap are: 1, sulfophenazole; flunixin; fenoprofen; norfloxacin. They are all antibacterial and anti-inflammatory drugs, which shows that galanthamine is also very likely to have certain anti-inflammatory efficacy.

TABLE 1 CMap P values top four drug profiles

Meanwhile, the combination of NGF + HotNet + CMap database analysis is adopted to predict the drug effect of galanthamine; the Network-Guided forms (NGF) uses the differential gene expression profile and the gene Network as input, and a classification model capable of correctly dividing the expression profile into corresponding conditions is obtained through training. NGF assigns an Importance Score (IS) to each gene and gene interaction in the gene network based on its contribution to NGF classification ability. The HotNet method is a combined method for processing up/down regulation data respectively based on a thermal diffusion theory, and can identify key sub-networks with significant differences in a gene interaction network. Downloading enteritis patient chip data from NCBI-GEO Dataset, wherein the chip number is as follows: GSE11223(202 samples), disease group as positive sample, and healthy group as negative sample, and calculate the importance score of 12929 genes in the chip by NGF. Selecting genes with the importance of 3000 top, inputting HotNet2 to construct enteritis disease pathogenic gene sub-network (using STRING-PPI) by taking the importance scores as initial heat; then, selecting a sub-network with the minimum P value of 0.28 from the obtained sub-networks, corresponding genes in the sub-networks to the probe of the CMap, determining the names of the probes which are up-down regulated according to the fold change value calculated by the chip, inputting the CMap, and searching for the drugs with similar expression profiles. As shown in fig. 4, among the top-ranked drugs with negative mean, nortriptyline can treat irritable bowel syndrome, and trichostatin a and roxarsone have antifungal and anti-insect effects, respectively, suggesting that galantamine may have anti-inflammatory effects on the intestinal tract.

TABLE 2 NGF + HotNet + CMap rank top 3 drug profiles

Example 2 application of galanthamine as feed additive in relieving soybean meal induced enteritis in zebra fish

1. Zebra fish SBMIE modeling and galanthamine addition experiment feed formula and preparation method

TABLE 3 Zebra fish SBMIE modeling and galanthamine addition experiment feed formula

FM is control fish meal feed, 50SBM is soybean meal feed (50% alternative protein source) used in this example, 50SBM +0.5ppm Gal is soybean meal feed supplemented with 0.5ppm galantamine, and 50SBM +1ppm Gal is soybean meal feed supplemented with 1ppm galantamine.

The preparation method comprises the following steps:

(1) sieving fish meal, bean pulp, starch, flour, cellulose, mineral premix, vitamin premix and the like with a 60-mesh sieve, wherein the fish meal, the bean pulp and the mineral mixture may contain larger particles such as fishbone, bean skin and the like, and sieving with the 60-mesh sieve after crushing; for less vitamin and mineral content: VD3, VK3, VB12, thiamine, VB6, folic acid, copper sulfate and sodium selenite need to be diluted by 20 times to prepare a premix; galanthamine needs 100 times diluted premix; said mineral mixture contains (g): magnesium sulfate (MgSO)₄·2H₂O) 60.530g, ferrous sulfate (FeSO)₄·H₂O)23.110g, copper sulfate (CuSO)₄·5H₂O)0.010g, zinc sulfate (ZnSO)₄·H₂O)0.620g, manganese sulfate (MnSO)₄·H₂O)1.640g, potassium iodide (KI)0.070 g, sodium selenite (NaSeO)₃)0.005g, adjusted to 1kg with starch; said vitamin premix comprises (g): vitamin B1(Thiamin)0.05, vitamin B2(Riboflavin)0.55, vitamin B6(pyridoxine)0.59, vitamin B12(cyanocobalamine)0.83, pantothenic acid (pantothenic acid) 2.89, folic acid (folic acid)0.40, inositol (inositol)19.39, niacin (niacin)2.24, biotin (biotin)4.91, vitamin c (ascorbic)7.16, vitamin a (vitamin a)2.40, vitamin d (vitamin d)0.40, vitamin e (vitamin e)12.55, vitamin k (vitamin k)0.80, adjusted to 1kg with starch;

(2) respectively weighing fish meal, bean pulp, starch, flour, cellulose, a mineral premix, a vitamin premix and a galanthamine premix according to the requirements of the weight percentages of the components in the feed formula in proportion;

(3) fully stirring and uniformly mixing the weighed fish meal, bean pulp, starch, cellulose and flour;

(4) adding the mineral premix, the vitamin premix and the galanthamine premix into the mixture obtained in the step (3), and continuously stirring to uniformly mix the mixture as much as possible;

(5) adding the fish oil into the mixture obtained in the step (4), fully stirring and uniformly mixing;

(6) adding a proper amount of purified water (10%) into the mixture obtained in the step (5) by using a watering can, continuously stirring, and uniformly mixing to obtain a mixture;

(7) preparing the mixture in the step (6) into granules with the particle size of 0.4-0.6mm by using a granulator; drying the prepared granules in a dryer at 58-60 deg.C until the water content is about 10%; oven drying, pulverizing, sieving with 15-35 mesh sieve to obtain granulated feed with corresponding specification, packaging, and storing in-20 deg.C refrigerator.

2. After all test zebra fishes (adult fishes of 2 months old) are fed with the fish meal group feed for 2 weeks, the corresponding groups of feeds are respectively fed for 3 weeks according to the groups, the feeding is slowly fed each time until the fishes do not eat the fish meal, the feed intake is about 23mg per fish per day, the growth and the development of the zebra fishes are normal in the test stage, and the survival rate is more than 98%.

MS-222 is used for anesthesia (the concentration is 0.1mg/ml), intestinal tract samples are dissected and taken, and the prevention and control effects are detected through qPCR, Western blot, H.E. staining and immunohistochemistry, and the results are as follows:

pathological section morphological level: in each group of zebra fish, including a fish meal group, a bean pulp and galanthamine 0.5ppm group and a bean pulp and galanthamine 1ppm group, a rear intestine tissue (n is 3) is fixed, the rear intestine tissue is subjected to gradient dehydration, wax immersion, paraffin embedding and slicing (5 microns), H.E. staining is carried out, morphological observation is carried out under a normal microscope under white light, and an experimental result (shown in figure 5) shows that 0.5-1ppm galanthamine is added into 50% of bean pulp substituted feed to prevent fish enteritis caused by the bean pulp, and the intestinal villus structure of the 1ppm group is better than that of the 0.5ppm group.

Growth level: growth indicators include total weight gain (weight gain), initial body weight (initial body weight), feed efficiency (feed efficiency), final body weight (final body weight), feed intake (feed intake), specific growth rate (specific growth rate); statistical analysis of growth indicators: the significance of the difference was obtained using mean and standard deviation analysis followed by t-test, with p <0.05 being significant and p <0.01 being very significant. The result shows (see fig. 6), from multiple growth indexes, the soybean meal feed added with galanthamine has the effect of helping fish bodies recover from growth inhibition caused by the soybean meal feed, and specifically shows that the growth inhibition can be relieved when the addition amount of 0.5ppm is added (although only the feed intake is obviously recovered, the final weight and the total weight gain are reduced), and the normal growth can be basically recovered when the addition amount of 1ppm is added (the final weight, the total weight gain, the feed intake and the feed efficiency are obviously recovered).

Gene expression level:

(1) level of transcription

Taking hindgut tissue (n ═ 3), extracting RNA, preparing a cDNA template, and then carrying out qPCR (quantitative polymerase chain reaction) to detect the expression quantity change of immune, tight junction and apoptosis genes at the transcription level, wherein the immune genes comprise important transcription factors NK-kb and Foxp3a for regulating immune response, enteritis related pattern recognition receptors TLR4, proinflammatory factors IL1b, TNF-alpha, chemotactic factors IL8, anti-inflammatory factors TGF-beta and IL10 (see figure 7A); the tight junction genes include ZO-1, occludin, claudin (1, b, c, h, 12, 15a) (see FIG. 8A); the apoptosis genes include caspase 3, caspase 8, caspase 9 (see FIG. 8B). The results show that: from the transcription level, the important transcription factor NF-kB for starting immune response, enteritis related pattern recognition receptor TLR4, acute inflammatory factor IL1b and TNF-a, chemotactic factor IL8 and immunoregulation related transcription factor Foxp3 (only 1ppm shows remarkable performance) can obviously reduce inflammatory high expression; and for the anti-inflammatory effect factors TGF-b and IL-10, inflammatory expression inhibition can be obviously reversed. From the protein level, the addition of 0.5ppm can reduce the high expression of Foxp3, IL17 and TNF-a caused by soybean meal feed, improve the expression level of an anti-inflammatory factor IL-10, and stimulate the expression of a helper T cell surface molecular marker CD4 in an intestinal villus lamina propria (LP layer) when the addition amount reaches 1ppm, which suggests that other Th cell groups start to play a role. From the transcription level of the compact junction gene, the inflammatory expression inhibition of the TJ junction gene ZO-1 and TJ strut genes ocludin, claudin 1, claudin b (only 1ppm shows remarkable) and claudin c is converted by adding galantamine, and meanwhile, the inflammatory high expression of calcium channel protein claudin 12 and potassium channel claudin 15a is remarkably reduced. From the transcriptional level of the apoptosis gene, the inflammatory overexpression of caspase 3, 8 and 9 can be obviously reduced.

(2) Protein level

Western blot detection of the change of the expression level of protein level immune genes, in particular to the preparation of a posterior intestinal tissue protein homogenate, and the Western blot detection of the change of the expression level of the protein level is carried out by adopting immune protein antibodies including Foxp3a related to immune regulation, a marker protein CD4 on the surface of an auxiliary T cell, a main proinflammatory factor IL17 of enteritis, and inflammation-inhibiting factors TNF-a and IL 10. The results show (see fig. 7B) that the intestine IL17, CD4 and Foxp3 were significantly reduced, while the anti-inflammatory factors TNF-a and IL10 were significantly increased after the galantamine-added soybean meal feed group fish compared to the soybean meal feed group without galantamine.

Immunohistochemical detection of the change in the localization of tissue cells expressed by the protein level of the immune gene is carried out by fixing the posterior intestinal tissue in 4% paraformaldehyde overnight (4 ℃), then embedding in OCT embedding medium for freezing at-20 ℃, carrying out frozen section (7 microns), carrying out immunohistochemical experiment by using CD4 antibody, and detecting the spatiotemporal change in the localization of proteins of the helper T cell surface antigen CD 4. The result shows that the addition of 0.5ppm galanthamine in the posterior intestinal tissue lamina propria can obviously reduce the number of CD4 positive cells, and the brightness of CD4 positive cells is recovered to be consistent with that of the fish meal group; whereas addition of 1ppm galantamine also significantly reduced CD4 positive cells, some CD4 positive cells were very bright suggesting that helper T cells of a different population than the inflammatory phase may be functioning.

By integrating the expression changes of the transcription level and the protein level, the galanthamine with the added concentration of 0.5-1ppm in the obtained feed has the effect of relieving the bean pulp induced enteritis on various aspects of intestinal tissue inflammation, including tissue morphology, gene expression (including immunity, tight junction, apoptosis) and the like, and can promote the recovery of a series of growth indexes of fish bodies.

The zebra fish adopted in the embodiment is a model fish recognized in the world at present, and belongs to cyprinid fishes. Freshwater fishes such as four Chinese carps which are liked by Chinese belong to cyprinid fishes, the cyprinid fishes have close genetic backgrounds and high gene homology, so that galanthamine is presumed to be suitable for preventing food-borne enteritis (mainly enteritis caused by soybean meal) of cyprinid fishes by modeling diseases of enteritis caused by the soybean meal of zebra fishes and detecting the expression of genes related to intestinal tissue morphology, growth indexes, immunity and intestinal mucosa barriers.

Example 3 intramuscular injection of galantamine to reduce the expression of inflammatory factors in the intestinal tract after induction of enteritis-affected grass carp by soybean meal

1. Taking 10 grass carps (the SBMIE modeling method and the feed formula of the grass carps refer to integrated transfer culture fresh animals Mechanism for the research to the society mean Stress in Fish Gut and live, and Frontier in Physiology) which are fed with 70% of Soybean Meal feed group and Fish Meal group for 7 weeks, keeping the temperature in a net cage at 23-25 ℃, and keeping the dissolved oxygen at more than 5mg/L, and fasting;

2. injecting galanthamine hydrobromide (0.05-0.1 mg/kg of fish weight) into the back muscle for 2 times every day for 2 days, and taking an intestinal tissue sample on the 4 th day;

3. and (3) carrying out fluorescence quantitative qPCR analysis to detect the expression condition of the immune genes related to the intestinal inflammation. The result of the statistical analysis of fluorescence quantitative data shows that compared with the diseased fish injected with PBS, galantamine injection can obviously reduce the expression level of immune genes related to acute inflammation in the intestinal tissues of SBMIE grass carp, including proinflammatory factor IL-1b, T cell surface marker CD4 and regulatory lymphocyte transcription factor Foxp3 (figure 9); meanwhile, the Th2 cell transcription factor T-beta associated with chronic inflammation is slightly increased, while STAT3 associated with autoimmunity is restored to the level similar to the fish meal group, and IL17 changes are not significant.

Example 4 evolutionary analysis of cholinesterase and acetylcholine receptor proteins of commonly-cultivated fish

First, to demonstrate whether the inhibitory effect of galantamine on cholinesterase is conserved in fish, we performed NJ tree evolution analysis on fish cholinesterase proteins (acting substrates of galantamine). The results of sequence search showed that cholinesterase protein is widely present in teleostean (see FIG. 10); in addition, cartilaginous fish also have cholinesterase proteins (see FIG. 10). The sequences, except the protein sequences of zebrafish and grass carp, were from EMSEMBL (http:// www.ensembl.org /) and grass carp genome respectively: (http://bioinfo.ihb.ac.cn/gcgd) The prediction of genome splicing in (2) and other fish and mammalian sequences are derived from NCBI. The results of the evolutionary analysis show that the cholinesterase protein sequences of fishes (including Danio reo, Cyprinus carpio, Oryzias latipes, Ctenopharyngodon idellus, Larimichthys crocea, Monopterus albus, Oreochromis niloticus, fascicularis giganteus) and mammals (human Homo sapiens and mouse Mus musculus) are well conserved and clustered into one branch. From the above results, it can be presumed that the action of galantamine as a cholinesterase inhibitor is evolutionarily conserved, and it acts not only on mammals but also on fish, and may have significant effects on both herbivorous (e.g., grass carp) and omnivorous fish (e.g., carp, zebrafish, medaka, tilapia, finless eel, large yellow croaker).

The effect of galantamine as a cholinesterase inhibitor, expressed as an increase in total choline in the body, must potentiate the activation of the cholinergic anti-inflammatory pathway by signaling through the acetylcholine receptor. Therefore, in order to demonstrate the extensive existence of an acetylcholine anti-inflammatory pathway in cultured fishes, protein sequence alignment and NJ tree evolutionary analysis construction are also carried out on the acetylcholine receptor nAChR-alpha7 of common cultured fishes. The acetylcholine receptor nAChR-alpha7 protein is in grass carp genome: (http://bioinfo.ihb.ac.cn/gcgd) Find 2 genes in the sequence and download their coding sequences. Other fish acetylcholine receptor nAChR-alpha7 protein sequences are derived from NCBI. The evolution analysis of the acetylcholine receptor nAChR-alpha7 protein in the cholinergic anti-inflammatory pathway in fish (see figure 11) shows that the receptor is possessed by common cultured fish, and the similarity of the protein amino acid sequence is high (80% -100%). Except that the grass carp nAChR-alpha7 gene comes from grass carp genome database of aquatic organism research institute of Chinese academy of sciences, the gene numbers are CI _ GC _10747 and CI _ GC _18333, and the predicted protein sequence is downloaded; other fish species (including zebrafish, carp, herring, Clupea harengus, Sinocyclocheilus grahami, Ictalurus punctatus, Monopterus albus, Pseudosciaena crocea, Tilapia mossambica, Atlantic salmon Salmo salar, Oncorhynchus mykiss, Arctic red salmon Salvinus alpinus), andthe human nAChR-alpha7 proteins are all from NCBI. Thus, it is presumed that various kinds of common food-feeding farmed fish have acetylcholine receptors necessary for the choline-activated anti-inflammatory pathway.

Therefore, by combining the above analysis of evolution of cholinesterase and choline receptors, it is presumed that galantamine is likely to have a protective effect against enteritis caused by soybean meal in cultured fishes having acetylcholine receptors (particularly herbivorous and omnivorous fishes). The fishes related to the invention specifically comprise fishes of the family Cyprinidae, such as grass carp, herring, silver carp, bighead carp, crucian carp, megalobrama amblycephala and the like, salmon and trout, such as Atlantic salmon, rainbow trout and the like, tilapia, channel catfish, finless eel, large yellow croaker and the like.

Claims (7)

1. The galanthamine can be used for preparing feed additive for relieving enteritis of cultured fish caused by soybean meal feed.

2. The galanthamine can be used for preparing medicines for preventing or treating enteritis of cultured fishes caused by soybean meal feed.

3. The use according to claim 1, wherein the farmed fish are herbivorous or omnivorous farmed fish.

4. The use according to claim 2, wherein the farmed fish are herbivorous or omnivorous farmed fish.

5. The use according to claim 1, wherein the farmed fish is cyprinus carpio.

6. Use according to claim 2, wherein the farmed fish is cyprinus carpio.

7. Use according to claim 2, wherein galantamine is injected in an amount of 0.05-0.1

mg/kg fish weight.

Images