Construction method and application of litopenaeus vannamei intestinal oxidative damage model

Abstract

The invention discloses a construction method and application of a litopenaeus vannamei intestinal oxidative damage model, and belongs to the technical field of animal model construction. The construction method comprises the step of feeding litopenaeus vannamei with the weight of 6-10g per litopenaeus vannamei with feed containing a malondialdehyde solution, the mass concentration of the malondialdehyde solution is 10%; the crude protein content of the feed is 40-42%, the crude fat content of the feed is 6-7%, and the peroxide value content of the feed is lower than 10.0 meq / kg. The content of malondialdehyde in the feed is 200 mg / kg; the feeding days are not less than 14 days. The litopenaeus vannamei intestinal oxidative damage model constructed by the invention can be used as a general model of the litopenaeus vannamei intestinal oxidative damage caused by grease oxidation, and has the advantages of short modeling time, simplicity and convenience in operation, good repeatability, typical pathological expression, no animal death and the like, so that the model becomes stable and reliable.

Description

Technical Field

[0001] The invention relates to the technical field of animal model construction, and in particular to a method for constructing an intestinal oxidative damage model of Litopenaeus vannamei and an application thereof. Background Art

[0002] Oils and fats provide energy and essential fatty acids for the growth of aquatic animals, and have an important impact on the growth, metabolism and feed costs of aquatic animals. However, during storage and processing, the non-conjugated double bonds of unsaturated fatty acids in feed oils and fats are highly unstable. Under the induction of heat, light, oxygen, etc., lipid peroxidation reactions are prone to occur, producing a series of harmful primary and secondary oxidation products such as aldehydes and ketones, causing oxidative damage to the tissues and organs of aquatic animals, destroying normal physiological functions, and endangering health. In shrimp farming and production, fish oil and other oils rich in unsaturated fatty acids are often used as feed raw materials. Oxidative damage to the shrimp intestine caused by oxidation of fish oil is very common in production. The resulting damage to the intestinal barrier function, decreased digestion ability and immunity have caused significant losses to aquaculture production.

[0003] Malondialdehyde is a classic end product of the oxidation of unsaturated fatty acids in oils and fats. It has good stability and is one of the sensitive indicators for objectively evaluating the degree of oil rancidity. Malondialdehyde has high biological activity and can cause lipid peroxidation in the intestinal epithelial cell membrane, thereby destroying the cell membrane structure and intracellular enzyme activity, inducing DNA damage and cell apoptosis, and then causing tissue damage. Studies have shown that many shrimps are sensitive to malondialdehyde in feed and can induce pathological reactions in a short period of time. However, the relevant model has not yet been established, resulting in a lack of effective tools for pathological and pharmacological studies of intestinal damage caused by oil oxidation. Summary of the invention

[0004] The purpose of the present invention is to provide a method for constructing an intestinal oxidative damage model of Penaeus vannamei and its application to solve the problems existing in the above-mentioned prior art. The present invention uses Penaeus vannamei as an experimental animal to construct an animal model of malondialdehyde-induced intestinal oxidative damage, providing an effective tool for pathological and pharmacological research on lipid oxidation-induced intestinal oxidative damage of shrimp.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] Technical solution 1: A method for constructing an animal model of malondialdehyde-induced intestinal oxidative damage, comprising the step of feeding feed containing malondialdehyde solution to Penaeus vannamei weighing 6-10 g / tail.

[0007] Since the age of shrimp is more difficult to determine than that of mammals, "weight" rather than "age in days" is usually used in production to indicate the growth stage of shrimp.

[0008] Furthermore, the mass concentration of the malondialdehyde solution is 10%.

[0009] Furthermore, the feed has a crude protein content of 40-42%, a crude fat content of 6-7% and a peroxide value content of less than 10.0 meq / kg.

[0010] Furthermore, the malondialdehyde content in the feed is 200 mg / kg.

[0011] Furthermore, the feeding days are not less than 14 days.

[0012] Furthermore, the weight of each of the Penaeus vannamei shrimp is 8-10 g.

[0013] Furthermore, the feeding frequency is 3 times a day.

[0014] Technical Solution 2: A method for evaluating the malondialdehyde-induced intestinal oxidative damage animal model constructed by the construction method, comprising the following steps:

[0015] A control group was set up, wherein the control group was fed with a feed without malondialdehyde, and the intestinal oxidative damage animal model induced by malondialdehyde was used as the model group. The following indicators were detected in the model group and the control group:

[0016] 1) Histopathological characteristics of the midgut;

[0017] 2) expression levels of genes related to midgut inflammation, tight junctions, and antioxidant enzyme activity;

[0018] The differences in indicators between the model group and the control group were used to confirm whether the intestinal oxidative damage animal model was successfully constructed.

[0019] Furthermore, the histopathological characteristics include tissue sections and fluorescence quantitative analysis.

[0020] Furthermore, the difference in indicators between the model group and the control group is used to confirm whether the intestinal oxidative damage animal model is successfully constructed: when compared with the control group, the villus height, villus width, intestinal wall thickness and muscle layer thickness of the model group are significantly decreased, with an average decrease of >25%; the relative expression levels of TNF-a, TGF-β1 and Nrf2 genes in the intestine of the model group are significantly increased, with an average increase of >80%; the relative expression levels of Keap-1, lL-2, ZO-1, dorsal, CAT and SOD genes in the intestine of the model group are significantly decreased, with an average decrease of >30%, then the intestinal oxidative damage animal model is successfully constructed.

[0021] Technical solution three: Application of the malondialdehyde-induced intestinal oxidative damage animal model constructed by the construction method in drug screening for lipid oxidation-induced intestinal oxidative damage in shrimp.

[0022] The present invention discloses the following technical effects:

[0023] Litopenaeus vannamei is one of the three major shrimp culture varieties in the world and is also the shrimp variety with the largest culture volume in my country. Its tissue structure and physiological characteristics are typical and representative among the species of the Penaeidae family, so the present invention selects Litopenaeus vannamei as the experimental animal for model construction. The Litopenaeus vannamei intestinal oxidative damage model constructed by the present invention can be used as a general model of shrimp intestinal damage caused by lipid oxidation, and has the advantages of short modeling time, simple operation, good repeatability, typical pathological manifestations, and no animal death, thereby making the model stable and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 This is a section of the midgut tissue of Penaeus vannamei (HE, cross section, 40X); A is the control group and B is the model group; the black arrow indicates the height of the villi, the yellow arrow indicates the width of the villi, the red arrow indicates the thickness of the intestinal wall, and the green arrow indicates the thickness of the muscular layer;

[0026] Figure 2 The height of intestinal villi, thickness of villi, thickness of intestinal wall and thickness of muscular layer in Litopenaeus vannamei. The results are expressed as mean ± standard error. * indicates that there is a significant difference between the control group and the model group (P<0.05).

[0027] Figure 3 Figure 2 Expression of genes related to intestinal inflammation, tight junctions and antioxidant enzyme activity in Litopenaeus vannamei. The results are expressed as mean ± standard error. * indicates significant difference between control group and model group (P<0.05). TNFα is tumor necrosis factor-α; TGF-β is transforming growth factor β; Nrf2 is nuclear erythroid 2-related factor; Keap-1 is tumor suppressor gene; lL-2 is interleukin 2; ZO-1 is tight junction protein; dorsal is antimicrobial peptide transcription factor; CAT is catalase; SOD is glutathione peroxidase. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] Example 1

[0034] The method for constructing an intestinal oxidative damage model of Litopenaeus vannamei comprises the following steps:

[0035] (1) Prepare a special pellet feed for Penaeus vannamei with a crude protein content of 40%, a crude fat content of 6%, and a peroxide value of 6.5 meq / kg (commercially available). Spray a 10% malondialdehyde solution (malondialdehyde (purity> 99%) dissolved in sterile water) evenly on the surface of the feed to make the malondialdehyde content in the feed reach 200 mg / kg, and dry it naturally before feeding.

[0036] (2) The shrimp Penaeus vannamei with an average body weight of 8.6±0.3g were divided into two groups for the experiment, with 3 replicates in each group, and were respectively raised in 6 aquariums of the recirculating culture system, each aquarium had a volume of 400L, and 30 shrimps were placed in each aquarium. The control group was fed with a control feed (a special pellet feed without malondialdehyde), and the model group was fed with a modeling feed (a special pellet feed for Penaeus vannamei prepared in step (1)). The control group and the model group were fed continuously for 15 days, 3 times a day until full. The midgut of the shrimp was taken for HE staining and pathological analysis of tissue sections, and the expression levels of genes related to midgut inflammation, tight junctions and antioxidant enzyme activity were detected by fluorescent quantitative PCR. The differences in indicators between the model group and the control group were used to confirm whether the intestinal oxidative damage animal model was successfully constructed: compared with the control group, the villus height, villus width, intestinal wall thickness and muscular layer thickness of the model group were significantly decreased; the relative expression levels of TNF-a, TGF-β1 and Nrf2 genes in the intestine of the model group were increased; the relative expression levels of Keap-1, lL-2, ZO-1, dorsal, CAT and SOD genes in the intestine of the model group were reduced, indicating that the intestinal oxidative damage animal model was successfully constructed.

[0037] The specific method of fluorescence quantitative PCR is as follows: the intestinal RNA is extracted using the TransZol Up Plus RNA Kit (TransGenBiotech), the concentration is measured by a micro-UV-Vis spectrophotometer NanoDrop One (Thermo Scientific), and then The One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech) was used to reverse the reaction into cDNA and stored in a -20°C refrigerator for later use. RT-qPCR was performed on a CFX Connect fluorescent PCR instrument using a PrefectStartTM Green qPCR SuperMix Kit (TransGen Biotech). The reaction system included: 1 μL cDNA, 0.4 μL each of upstream and downstream primers, 10 μL of 2xPerfectStartTM Green qPCR SuperMix, 0.4 μL of Passive Reference Dye, and 7.8 μL of Nuclease-free Wter. The amplification conditions were: preheating at 94°C for 30 seconds, denaturation at 94°C for 5 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 10 seconds, for a total of 40 cycles. The results were analyzed using Calculate the relative expression of the target gene.

[0038] (3) Optical microscopy results showed that compared with the control group, the model group had pathological features such as disordered arrangement of midgut epithelial cells, detachment of intestinal epithelial cells from the basement membrane, damage and shedding of some intestinal epithelial microvilli, necrosis of some cells, decreased height and width of villi, and thinning of intestinal wall thickness and muscle layer thickness ( Figure 1 ).

[0039] Compared with the control group, the villus height, villus width, intestinal wall thickness and muscle thickness of the model group decreased by 26%, 52%, 15% and 30% (P<0.05), with an average decrease of 31% ( Figure 2 ).

[0040] Compared with the control group, the relative expression levels of TNF-a, TGF-β1, and Nrf2 genes in the intestine of the model group increased by 108%, 58%, and 154% (P<0.05), with an average increase of 107%; the relative expression levels of Keap-1, lL-2, ZO-1, dorsal, CAT, and SOD genes decreased by 52%, 49%, 54%, 33%, 35%, and 44% (P<0.05), with an average decrease of 45% ( Figure 3 ). This showed that the model group induced inflammation in midgut cells, leading to a decrease in cellular immunity and antioxidant capacity, and the destruction of tight junctions.

[0041] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing an animal model of intestinal oxidative damage induced by malondialdehyde, characterized in that: The method comprises the step of feeding feed containing malondialdehyde solution to Litopenaeus vannamei with a body weight of 6-10g / tail.

2. The construction method according to claim 1, characterized in that: The mass concentration of the malondialdehyde solution is 10%.

3. The construction method according to claim 1, characterized in that: The feed has a crude protein content of 40-42%, a crude fat content of 6-7% and a peroxide value content of less than 10.0 meq / kg.

4. The construction method according to claim 1, characterized in that: The malondialdehyde content in the feed is 200 mg / kg.

5. The construction method according to claim 1, characterized in that: The number of days for throwing something and feeding is not less than 14 days.

6. The construction method according to claim 1, characterized in that: The feeding frequency is 3 times a day.

7. A method for evaluating an animal model of intestinal oxidative damage induced by malondialdehyde constructed by the construction method according to any one of claims 1 to 6, characterized in that: The following steps are involved: A control group was set up, wherein the control group was fed with a feed without malondialdehyde, and the intestinal oxidative damage animal model induced by malondialdehyde was used as the model group. The following indicators were detected in the model group and the control group: 1) Histopathological characteristics of the midgut; 2) expression levels of genes related to midgut inflammation, tight junctions, and antioxidant enzyme activity; The differences in indicators between the model group and the control group were used to confirm whether the intestinal oxidative damage animal model was successfully constructed.

8. The evaluation method according to claim 7, characterized in that: The histopathological characterization includes tissue sectioning and fluorescence quantitative analysis.

9. The evaluation method according to claim 7, characterized in that: The difference between the indicators of the model group and the control group is used to confirm whether the intestinal oxidative damage animal model is successfully constructed: when compared with the control group, the villus height, villus width, intestinal wall thickness and muscle layer thickness of the model group are significantly decreased, with an average decrease of >25%; the relative expression levels of TNF-a, TGF-β1 and Nrf2 genes in the intestine of the model group are significantly increased, with an average increase of >80%; the relative expression levels of Keap-1, lL-2, ZO-1, dorsal, CAT and SOD genes in the intestine of the model group are significantly decreased, with an average decrease of >30%, and the intestinal oxidative damage animal model is successfully constructed.

10. Use of an animal model of malondialdehyde-induced intestinal oxidative damage constructed by the construction method according to any one of claims 1 to 6 in screening drugs for lipid oxidation-induced intestinal oxidative damage in shrimp.

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