CN113040291A

CN113040291A - Application of PPAR gamma activator in improving environmental stress resistance of aquatic animals

Info

Publication number: CN113040291A
Application number: CN202110373554.6A
Authority: CN
Inventors: 乔芳; 王雪; 杜震宇
Original assignee: East China Normal University
Current assignee: East China Normal University
Priority date: 2021-04-07
Filing date: 2021-04-07
Publication date: 2021-06-29

Abstract

The invention discloses an application of a PPAR gamma activator in relieving lipotoxicity of aquatic animals and improving the capability of resisting environmental stress of the aquatic animals.

Description

Application of PPAR gamma activator in improving environmental stress resistance of aquatic animals

Technical Field

The invention relates to the field of aquaculture, in particular to application of a PPAR gamma activator in relieving lipotoxicity of aquatic animals and improving the capability of resisting environmental stress of the aquatic animals.

Background

The environmental stress refers to environmental stimulation which can cause the animal body to present a "tension state" in the environment, and the common environmental stress comprises low temperature, high temperature, water quality deterioration and the like. The response of fish to environmental stress is a non-specific physiological response. Environmental stress is not a disease in itself, but it is a cause of one or more diseases. Various environmental stresses are generally suffered in the cultivation process of aquatic animals such as fishes, and the aquatic animals are seriously damaged or even die due to the excessive or overlong environmental stresses, which causes serious losses to breeders. Therefore, the method has strong practical value for improving the resistance of aquatic animals such as fishes to environmental stress.

In order to enhance the resistance of aquatic animals to environmental stress and improve the culture yield, the method generally adopts the modes of adding the artificial treatment of river water into a culture pond, periodically disinfecting the water body, adding vitamin C into feed and the like.

Further, peroxisome proliferator-activated receptor gamma (PPAR γ) is a key transcriptional regulator of adipogenesis, and can directly regulate the expression of genes related to adipocyte differentiation and lipid metabolism.

However, as a result of no study, PPAR γ has been associated with the resistance of aquatic animals such as fish against environmental stress, and it is unknown whether or not there is an interaction relationship between them.

Disclosure of Invention

In view of the above technical problems, the present invention provides an application of PPAR γ activator for improving the resistance of aquatic animals against environmental stress, which is to administer PPAR γ activator to aquatic animals (such as fish) to promote fat synthesis, reduce the level of free fatty acid in liver, thereby improving and relieving lipotoxicity, and improving the resistance of aquatic animals (such as fish) under stress conditions.

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

in one aspect, there is provided the use of a PPAR γ activator in the manufacture of a composition for enhancing the ability of an aquatic animal to resist environmental stress, and the composition includes a PPAR γ activator.

Preferably, the PPAR γ activator is contained in an amount of 10-30mg/kg of the composition.

Preferably, the composition is a pharmaceutical composition or an aquatic feed additive or an aquatic animal health product.

Preferably, the composition further comprises a pharmaceutically or feedstuffs or animal health acceptable carrier or excipient.

Preferably, the carrier or excipient is an oleaginous base or a water-soluble base.

Preferably, the PPAR γ activator comprises one or more of rosiglitazone, ciglitazone, bezafibrate and clofibrate.

Preferably, the aquatic animals include fish, and more preferably, the fish is ornamental fish such as zebra fish (commonly called "striped fish"), ribbon medaka (commonly called: peacock fish), red crucian, immortal fish, etc.;

preferably, the improvement of the resistance of the aquatic animals to environmental stress comprises one or more of promotion of differentiation of fat cells, reduction of free fatty acid level, reduction of oxidative stress, improvement of resistance to cold stress, improvement of resistance to heat stress and improvement of resistance to ammonia nitrogen stress.

In one aspect, a composition is provided that can be administered to an aquatic animal and includes a PPAR γ activator.

Also provided is a method for constructing a zebrafish (Danio rerio) model for improving the ability of aquatic animals to resist environmental stress, comprising the steps of:

s1, knocking out a peroxisome proliferator-activated receptor gamma (PPAR gamma) gene of the zebra fish to obtain mutant zebra fish;

s2, breeding wild zebra fish and mutant zebra fish, detecting tissue samples of the wild zebra fish and the mutant zebra fish, and detecting the resistance of the wild zebra fish and the mutant zebra fish under stress reaction;

s3, dividing wild zebra fish into 2 groups serving as a wild control group and a wild experimental group respectively, and taking mutant zebra fish serving as a mutant experimental group, wherein the wild experimental group and the mutant experimental group are fed with a composition containing a PPAR gamma activator;

and S4, taking tissue samples of the zebra fish of the wild control group, the wild experimental group and the mutant experimental group for detection, and detecting the resistance of each group of zebra fish under stress reaction.

Preferably, the tissue sample detection indicators include: one or more of liver triglyceride, liver free fatty acid and the like, and the resistance detection indexes under stress reaction comprise: survival rate under cold stress, survival rate under heat stress and survival rate under ammonia nitrogen stress.

The invention has at least the following beneficial effects:

the invention improves and relieves lipotoxicity and improves the resistance of aquatic animals (such as fishes) under stress conditions by giving PPAR gamma activators to the aquatic animals (such as fishes) to promote fat synthesis and reduce the level of free fatty acid in the liver.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 shows the results of an experiment for evaluating the safety of PPAR γ activator (rosiglitazone);

FIG. 2 is a graph showing the triglyceride content, free fatty acid level and survival rate under cold stress, heat stress and ammonia nitrogen stress in the liver of zebra fish in example 1 after rosiglitazone was added to the composition at 10mg/kg, 20mg/kg and 30mg/kg, respectively;

FIG. 3 is a schematic diagram of the CRISPR/Cas9 technology-based zebra fish PPAR γ gene knockout in example 1;

FIG. 4 is a graph showing triglyceride content, free fatty acid level and survival under cold stress, heat stress, ammonia nitrogen stress in livers of wild type zebra fish and mutant zebra fish in example 2;

FIG. 5 is a graph showing triglyceride content, free fatty acid level and survival under cold stress, heat stress and ammonia nitrogen stress in livers of wild-type control group zebra fish, wild-type experimental group zebra fish and mutant experimental group zebra fish in example 2.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1:

the embodiment provides application of a PPAR gamma activator in preparing a composition for improving the capability of resisting environmental stress of aquatic animals, wherein the composition comprises the PPAR gamma activator and a carrier or excipient which is acceptable in pharmacy or composition science or animal health care science, and 10-30mg of the PPAR gamma activator is contained in each kg of the composition.

And providing a composition that can be administered to an aquatic animal and contains 10-30mg of a PPAR γ activator per kg of the composition.

The PPAR gamma activator comprises one or more of rosiglitazone, ciglitazone, bezafibrate and clofibrate, and the aquatic animals comprise fish, more preferably the fish is ornamental fish.

Meanwhile, the composition can be in the form of a pharmaceutical composition (including a vaccine) or an aquatic product composition additive or an aquatic animal health product, and the carrier or excipient is an oleaginous base or a water-soluble base.

The term "pharmaceutically or compositionally or animal nutraceutically acceptable carrier or excipient" refers to a carrier for the administration of a therapeutic agent, including various diluents, which are not essential active ingredients per se, and which are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art.

PPAR γ activator (rosiglitazone) safety assessment experiment:

control compositions were prepared according to the formulations shown in table 1.

TABLE 1 control composition formula

Composition formula (g/kg)	Control
		Casein as Casein	300
Gelatin	50
		Corn starch Corn starch	350
Soybean oil	70
		Vitamin premix1	4
Mineral premix2	8
		Choline chloride	5
2, 6-di-tert-butyl-4-methylphenol BHT	0.2
		Dimethyl-beta-propionic acid thetin (DMPT)	1
Calcium dihydrogen phosphate Ca (H)₂PO₄)₂	10
		Sodium carboxymethylcellulose CMC	40
Cellulose	161.8
		Total up to	1000

When preparing the experimental composition, rosiglitazone was first dissolved in dimethyl sulfoxide (DMSO) at a weight ratio of DMSO to soybean oil in the above formulation of 1:9, so the amount of DMSO required was calculated from the amount of soybean oil required in the above formulation. Mixing the DMSO in which the rosiglitazone is dissolved with the soybean oil uniformly, mixing the mixture with other components in the formula uniformly, pressing the mixture into strips, airing the strips, crushing the strips, sieving the strips, bagging the strips, and refrigerating the strips to obtain the experimental group composition with the addition amount of the PPAR gamma activator of 20 mg/kg.

The safety evaluation experiment is provided with a control group and an experiment group, each treatment group is provided with 3 parallel treatment groups, 6 transparent glass jars with the volume of about 30L are prepared, and 36 zebra fish are randomly placed in each glass jar.

Before starting the experiment, the initial total body weight of zebrafish per jar was recorded, and the control group was then fed with the composition of the experimental group (i.e. "20 mg/kg" group in fig. 1), which was fed 4-6% of body weight each day, twice a day in the morning and evening (9:00 and 17: 00). Weighing once a week, recalculating daily feeding amount according to body weight, and culturing for four weeks for each group.

During the culture experiment, excrement at the bottom of the tank is sucked away every two days, half of water is replaced, tap water subjected to aeration dechlorination is used for replacing water, the water temperature is kept at 26 +/-5 ℃, the pH value is 7.8, dissolved oxygen is higher than 6.5mg/L, and the 14-hour illumination and 10-hour light cycle at night are maintained. 3 zebra fish (total 6) are respectively taken from the experimental group and the control group and respectively placed in 6 small fish tanks (1 tail in one small tank) with the same size and specification, then the 6 small fish tanks are placed in a behavior instrument together, and the motion trail of each zebra fish is recorded at the same time.

After the culture is finished, 3 zebra fishes are randomly taken from 3 parallel jars of a control group and an experimental group respectively, and 9 zebra fishes are taken from each treatment group. The zebra fish is placed in 20mg/L MS-222 aqueous solution for anesthesia, the liver is collected, the livers of 2-3 zebra fish are mixed into a tube, 4 treatment groups are arranged in parallel, and the zebra fish is preserved at the temperature of minus 80 ℃. Detecting the expression level of apoptosis and inflammation related genes of zebra fish of each treatment group by using Nanjing construction kit, wherein the apoptosis and inflammation related genes comprise: caspase3a (caspase-3 a), bcl2(B cell lymphoma/leukemia-2), il1B (human interleukin 1. beta.), nf- κ B (nuclear factor κ B).

As can be seen from fig. 1, the composition of the experimental group to which 20mg/kg rosiglitazone was added did not affect the average weights of zebrafish at 2 weeks and 4 weeks, and did not significantly affect the expression levels of the 4 genes associated with apoptosis and inflammation, and at the same time, as can be seen from the movement trace of zebrafish, the zebrafish in the control group and experimental group did not have a significant difference in total distance of 5 minutes movement, thereby indicating that the addition of 20mg/kg rosiglitazone to other groups did not affect the growth and movement of zebrafish, and fully indicating that the administration of PPAR γ activators such as zebrafish rosiglitazone is safe.

The effect of the addition of PPAR γ activator (rosiglitazone) on the ability of zebrafish (Danio reio) to resist environmental stress is illustrated below in connection with the examples.

1) Preparation of control and Experimental compositions

Control compositions were prepared according to the formulations shown in table 1 above.

Also, with reference to the contents of the above safety evaluation test section, compositions having PPAR γ activator additive amounts of 10mg/kg (test group composition 1), 20mg/kg (test group composition 2) and 30mg/kg (test group composition 3) were obtained, respectively.

2) Starting a culture experiment, and collecting the liver of the zebra fish after the culture is finished

The breeding experiment comprises four treatment groups, namely a control group, an experiment group 1, an experiment group 2 and an experiment group 3, wherein each treatment group is provided with 3 parallel treatment groups, 12 transparent glass jars with the volume of about 30L are prepared, and 36 zebra fish are randomly placed into each glass jar.

Before the beginning of the culture, the initial total body weight of each jar of zebra fish is recorded, and then the control group is fed with the control group composition, and the experiment group 1, the experiment group 2 and the experiment group 3 are correspondingly fed with the experiment group composition 1, the experiment group composition 2 and the experiment group composition 3, wherein the control group composition is fed according to 4-6% of the body weight every day, and the zebra fish are fed twice in the morning and evening every day (9:00 and 17: 00). Weighing once every two weeks, recalculating daily feeding amount according to the weight, and culturing all the groups for four weeks.

During the culture period, the feces at the bottom of the tank are sucked away every two days, half of the water is replaced, the water is replaced by tap water after aeration and dechlorination, the water temperature is kept at 26 +/-5 ℃, the pH value is 7.8, the dissolved oxygen is higher than 6.5mg/L, and the 14-hour illumination and 10-hour light cycle at night are maintained.

After the culture is finished, 3 zebra fishes are randomly taken from 3 parallel tanks of a control group, an experimental group 1, an experimental group 2 and an experimental group 3 respectively, and 9 zebra fishes are taken from each treatment group. The zebra fish is placed in 20mg/L MS-222 aqueous solution for anesthesia, the liver is collected, the livers of 2-3 zebra fish are mixed into a tube, 4 treatment groups are arranged in parallel, and the zebra fish is preserved at the temperature of minus 80 ℃. The content of triglyceride and the concentration of free fatty acid in the livers of the zebra fish of each treatment group are measured by a Nanjing constructed kit.

3) Respectively carrying out cold stress, heat stress and ammonia nitrogen stress

Before cold stress treatment, 12 glass jars are placed into a biochemical incubator, each 3 glass jars are used as a treatment group, 4 treatment groups are totally placed and marked, each glass jar is adjusted to be in a 15 ℃ refrigeration mode, and then 10 zebra fishes are respectively taken from each glass jar of each treatment group and are respectively placed into corresponding small jars which are marked.

The experiment was started, the number of dead fish was counted every 12h, the survival rate was calculated and reduced by 1 ℃ until all zebrafish dead light in two or more pots in 3 parallel pots of any treatment group or the survival rate had significantly differed by 72 hours after the experiment was performed. Bait feed was stopped throughout the experiment.

Before heat stress treatment, the water bath is adjusted to 27 ℃, and the water bath is put into 12 small isolation tanks (plastic fish tanks with small holes on the periphery for facilitating water flow), and each 3 small isolation tanks are used as a treatment group, and 4 treatment groups are marked. And respectively taking 10 zebra fish from each glass jar of each treatment group, and respectively putting the zebra fish into corresponding marked small isolation jars.

The experiment was started, the temperature was raised by 1 ℃ every half hour until the temperature was raised to 38 ℃, then the water temperature of 38 ℃ was maintained, the number of dead fish was counted every 12h, and the survival rate was calculated until two or more zebra fish in 3 parallel vats of any treatment group all died, or the survival rate after the experiment was run for 72 hours had a significant difference. Bait feed was stopped throughout the experiment.

The ammonia nitrogen stress is that ammonium chloride (analytically pure) is used as an ammonia nitrogen source, the nitrogen concentration determined in the experiment is 85mg/L, namely about 648.85mg of ammonium chloride is added into glass jars with 2L of dechlorinated tap water, 12 glass jars are prepared according to the method, each 3 glass jars are used as a treatment group, 4 treatment groups are provided, 2L of dechlorinated tap water is filled in each glass jar, about 648.85mg of ammonium chloride is added into the tap water, and the treatment groups are marked.

At the beginning of the experiment, 10 zebra fish were taken from each glass jar of each treatment group and put into the 12 marked glass jars with 85mg/L nitrogen concentration.

Counting the number of dead fish every 12h, and calculating the survival rate until two or more zebra fish in 3 parallel vats of any treatment group die completely or the survival rate after the experiment is carried out for 72 hours has a significant difference. During the experiment, the test solution was changed every 24 hours by 50% of the total volume. Bait feed was stopped throughout the experiment.

4) Result calculation analysis

The content of triglyceride in liver and the concentration of free fatty acid are calculated according to a formula given in Nanjing constructed kit instructions, graph pad software is input for drawing after the calculation is completed, SPSS software is used for analyzing whether significant difference exists, the significant difference is set as P <0.05, and the very significant difference is set as P < 0.01.

After the survival data were calculated, the data were also entered into GraphPad software for mapping and analyzed for significant differences using SPSS software.

Survival rate at each time point is the number of live fish/initial total fish at the current time point.

As can be seen from FIG. 2(A), compared with the control group, the addition of 10mg/kg and 20mg/kg of rosiglitazone in the composition can significantly increase the content of triglyceride in the liver of zebra fish, and the effect of adding 20mg/kg of rosiglitazone is more obvious.

As can be seen from fig. 2(B), the addition of rosiglitazone to the composition was helpful to reduce the liver free fatty acid level of zebra fish compared with the control group, and the difference was significant when 20mg/kg of rosiglitazone was added.

As can be seen from fig. 2(C), the addition of rosiglitazone at 10mg/kg and 20mg/kg to the composition significantly improved the survival rate of zebrafish treated for 36 hours and 48 hours under cold stress, compared to the control group, wherein the effect of adding rosiglitazone at 20mg/kg was more significant.

As can be seen from fig. 2(D), the survival rate of zebrafish under heat stress for 60 hours can be significantly improved by adding 20mg/kg rosiglitazone to the composition; in addition, under the condition of heat stress for 72 hours, the survival rate of the zebra fish can be improved by adding 10mg/kg and 20mg/kg of rosiglitazone.

As can be seen from FIG. 2(E), the survival rates of the zebra fish under ammonia nitrogen stress for 60 hours and 72 hours can be remarkably improved by adding 20mg/kg of rosiglitazone.

Therefore, the principle that the addition of PPAR-gamma activators such as rosiglitazone and the like in the composition can relieve lipotoxicity and improve the resistance of the zebra fish to cold stress, heat stress and ammonia nitrogen stress can be fully demonstrated as follows:

PPAR-gamma is a ligand-dependent transcription factor, and PPAR-gamma activators such as rosiglitazone bind to the ligand binding domain of PPAR gamma and steroid receptor co-activator-1 (SRC-1) to form a ternary complex, thereby activating PPAR gamma. After activated, PPAR gamma can induce the expression of a plurality of genes related to lipid synthesis and promote the expression of CCAAT enhancer binding protein alpha (C/EBP alpha), and also can induce the expression of genes related to triglyceride uptake and storage, thereby promoting the differentiation of fat cells. In addition, PPAR γ can enhance the expression of lipoprotein lipase and free fatty acid transporters, such as fatty acid transporters and fatty acid translocators, thereby promoting the uptake of free fatty acids released from circulating lipoproteins, activating antioxidant response elements and antioxidant-related gene expression, and maintaining the redox homeostasis of cells.

It has been reported that elevated levels of free fatty acids, especially saturated free fatty acids, may play a key role in lipotoxicity mechanisms. If the excess free fatty acids are unable to synthesize triglycerides, the level of free fatty acids in the cells will increase, which can lead to lipotoxicity and oxidative stress, affecting the metabolic balance of the body.

After PPAR gamma is activated by activator such as rosiglitazone, on one hand, the PPAR gamma can promote the differentiation of preadipocytes and the intake of free fatty acid, and finally store the free fatty acid in the form of triglyceride, on the other hand, the PPAR gamma can reduce oxidative stress, thereby relieving the lipotoxicity of the organism. Lipotoxicity is known to produce oxidative stress, and environmental stress leads to cell or animal death associated with oxidative stress, and reducing oxidative stress can improve resistance of cells or animals to stress.

Example 2:

the embodiment provides a method for constructing a zebrafish (Danio rerio) model for improving the environmental stress resistance of aquatic animals, which comprises the following steps:

s1, knocking out a peroxisome proliferator-activated receptor γ (PPAR γ) gene of zebra fish to obtain a mutant zebra fish, in this embodiment, the PPAR γ gene of zebra fish is mainly knocked out based on CRISPR/Cas9 technology, which specifically includes the following steps:

the CRISPR/Cas9 target is designed by using an online tool ZiFiT target Version 4.2, the target sequence is GGGCTTCGGCCTGAGCGCTC, and a sequence is added before and after the target: TAATACGACTCACTATAGGGGCTTCGGCCTGAGCGCTCGTTTTAGAGCTAGAAATAGC, constructing CRISPR/Cas9 target sequence, and connecting the CRISPR/Cas9 target sequence into sgRNA (single guide RNA) plasmid;

and (2) performing PCR amplification by using the sgRNA plasmid as a template and the sgRNA-R as a downstream primer, wherein the PCR amplification system is as follows: template 1. mu.l, 10 XBuffer 5. mu.l, F1 primer (upstream primer, obtained by conventional primer design software) 2. mu.l, R1 primer (downstream primer) 2. mu.l, dNTP 5. mu.l, Taq enzyme 0.5. mu.l, ddH₂O34.5. mu.l, 50. mu.l in total;

the PCR amplification procedure was as follows: 94 ℃ for 5min, 94 ℃ for 30s, 56 ℃ for 30s, 72 ℃ for 15s (35 cycles), 72 ℃ for 5min, 4 ℃ for 1 h;

obtaining gDNA (genomic DNA) by PCR amplification, tapping and recovering, and purifying the gDNA;

transcription is carried out by using a Thermo T7 high-yield transcription kit, and the transcription system is as follows: NTP 8. mu.l (each 2. mu.l), 5 XBuffer 4. mu.l, gDNA template 5.5. mu.l, T7 Enzyme 2. mu.l, RNase inhibitor 0.5. mu.l, total 20. mu.l to obtain gRNA;

the transcription program is: placing in water bath at 37 ℃ for 6 h; b. adding 1.5 mul DNase into each tube, mixing uniformly, and carrying out water bath at 37 ℃ for 15 min;

after transcription is finished, 30 mu l of enzyme-free water and 31 mu l of 8M lithium chloride are added into each EP tube, the mixture is uniformly mixed, precipitation is carried out overnight at the temperature of minus 20 ℃, gRNA is precipitated, and the gRNA is washed by 75% ethanol and then dried;

dissolving gRNA with enzyme-free water, mixing gRNA with Cas9 protein (prepared now), and injecting zebra fish embryo by the following injection system: gRNA 0.2 μ l, Cas9 protein 0.5 μ l, Cas9 Buffer 0.3 μ l, total 1 μ l, gRNA final concentration of 400ng/μ l;

when injecting embryo, the injection pressure is adjusted to 20-40; laying roe, and adding needle 1.5 μ l; the injection volume (bleb) was egg 1/10 size; injection as close to the animal spine as possible;

designing a primer on NCBI according to a target point, wherein the primer sequence of PPAR gamma (transcript number: NM-131467) is F: CAACTGCAGATACATGCCGC, R: TGGTAGCTGTGGAAGAAGCG; after PCR amplification, SDS gel running or sequencing is carried out by a company, and F0-generation zebra fish with mutation is screened;

mating F0-generation zebra fish carrying mutation with wild zebra fish to generate F1 generation, screening out the mutation of the same type in F1 generation, mating male and female to obtain F2-generation zebra fish, screening out homozygote from F2-generation zebra fish, adding 6 base pairs on a target site of the homozygote and deleting 2 non-adjacent base pairs, and enabling PPAR gamma of the zebra fish to encode protein of 527 amino acids; however, the translation product of the gene knockout line with the premature stop codon only contains 72 amino acid residues, the PPAR gamma gene knockout target is shown in figure 3, and finally, the F2 generation homozygous zebra fish is subjected to male and female mating to obtain a large number of F3 generation zebra fish, namely, mutant zebra fish with the PPAR gamma gene knockout, namely F3 generation zebra fish;

s2, breeding the wild zebra fish and the mutant zebra fish for the same time under the same condition, and then respectively taking tissue samples of the wild zebra fish and the mutant zebra fish to detect and detect the resistance of the wild zebra fish and the mutant zebra fish under stress reaction; preferably, the tissue sample detection indicators include: one or more of liver triglyceride, liver free fatty acid and the like, and the resistance detection under stress reaction comprises the following steps: one or more of survival rate under cold stress, survival rate under heat stress and survival rate under ammonia nitrogen stress; the index detection and the resistance detection under stress reaction are the same as in example 1, and are not described again;

as shown in fig. 4, compared with wild zebra fish, the liver triglyceride level of the mutant zebra fish is significantly reduced, and the free fatty acid level of the liver is significantly increased, so that liver lipotoxicity is caused, and meanwhile, compared with wild zebra fish, the resistance of the mutant zebra fish to cold stress, heat stress and ammonia nitrogen stress is significantly reduced, so that the resistance of the fish to environmental stress is significantly related to lipotoxicity caused by imbalance of fat metabolism of the fish for the first time;

s3, setting three processing groups: the method comprises the following steps of (1) setting N (such as 3) parallel treatment groups in each treatment group, preparing 3 x N culture containers with the same specification, randomly putting M (such as 30) zebra fish into each culture container of the wild type control group, randomly putting M zebra fish into each culture container of the wild type experimental group and randomly putting M zebra fish into each culture container of the mutant type experimental group, wherein N and M are positive integers and can be set according to experimental needs;

recording the initial total body weight of each zebra fish, and then feeding a control group composition (such as a composition prepared by a formula in table 1) to a wild-type control group, and feeding an experimental group composition added with a PPAR gamma activator to both the wild-type experimental group and the mutant experimental group, wherein the PPAR gamma activator is added in an amount of 10-30mg/kg in the experimental group composition, in this embodiment, the experimental group composition is one or more of the experimental group composition 1, the experimental group composition 2 and the experimental group composition 3, preferably, the PPAR gamma activator is added in the same amount in the experimental group compositions fed to the wild-type experimental group and the mutant experimental group, and the amounts of the PPAR gamma activator in the experimental group compositions are 20 mg/kg;

feeding each treatment group according to 4-6% of the weight every day, feeding twice (9:00 and 17: 00) in the morning and evening every day, weighing once every two weeks, recalculating daily feeding amount according to the weight, and culturing for four weeks;

and S4, taking tissue samples of the zebra fish of the wild control group, the wild experimental group and the mutant experimental group for detection, and detecting the resistance of each group of zebra fish under stress reaction, wherein the detection indexes of the tissue samples and the detection indexes of the resistance under stress reaction are the same as those of S2, and are not repeated here, and the detection result is shown in FIG 4.

As can be seen from fig. 5(a) - (B), compared to the wild-type control group, the wild-type experimental group has significantly increased hepatic triglyceride content and significantly decreased hepatic free fatty acid level, while the mutant experimental group has significantly decreased hepatic triglyceride content and significantly increased hepatic free fatty acid level, because the mutant experimental group lacks PPAR γ gene in zebrafish, and cannot relieve lipid toxicity even though PPAR γ activator is added, while the wild-type control group and the wild-type experimental group have the opposite results to those in fig. 4, that is, the PPAR γ activator is added to the composition to increase hepatic triglyceride and simultaneously decrease hepatic free fatty acid level, which just proves that the PPAR γ activator can effectively relieve lipid toxicity.

Further, as can be seen from fig. 5(C), at 60 hours of cold stress, the survival rate of the wild type experimental group was significantly increased and the survival rate of the mutant type experimental group was significantly decreased compared to the wild type control group, and at 24, 36 and 48 hours of cold stress, the survival rate of the wild type experimental group was more increased and the survival rate of the mutant type experimental group was less decreased compared to the wild type control group; as can be seen from fig. 5(D), the survival rates at 60 hours of heat stress of the wild-type experimental group were significantly increased and at 36 and 48 hours of heat stress of the wild-type control group, while the survival rates at 48 and 60 hours of heat stress of the mutant experimental group were significantly decreased and at 12, 24 and 36 hours of heat stress of the mutant experimental group were decreased, as compared with the wild-type control group; as can be seen from fig. 4(E), compared with the wild-type control group, the survival rate of the wild-type experimental group under the ammonia nitrogen stress for 60 hours is greatly increased, and the survival rates under the ammonia nitrogen stresses for 24 hours, 36 hours and 48 hours are increased; compared with a wild control group, the survival rate of the mutant experimental group is obviously reduced under the ammonia nitrogen stress of 48 hours and 60 hours, and the survival rate under the ammonia nitrogen stress of 24 hours and 36 hours is in a descending trend. The result trend is similar to triglyceride and liver free fatty acid, that is, the survival rate of the zebra fish under the stress condition cannot be improved even if the PPAR gamma activator is added due to the lack of the PPAR gamma gene in the zebra fish of the mutant experimental group, while the resistance under the stress condition can be improved by the wild experimental group through the addition of the PPAR gamma activator.

Therefore, as can be seen from the results in fig. 5, the PPAR γ gene is closely related to the resistance of fish to environmental stress and lipotoxicity caused by imbalance of fat metabolism, and the addition of the PPAR γ activator in the composition can significantly improve and relieve the lipotoxicity, improve the resistance of fish under stress conditions, and improve the model building success of the zebra fish model with the capability of aquatic animals to resist environmental stress.

In conclusion, the invention discovers for the first time that the PPAR gamma gene is closely related to the resistance of aquatic animals such as fish to environmental stress and lipotoxicity caused by imbalance of fat metabolism, and the PPAR gamma activator is added into the composition to promote fat synthesis and reduce the level of free fatty acid in liver, so as to improve and relieve the lipotoxicity and improve the resistance of the aquatic animals such as fish under stress conditions.

The technical features of the above embodiments 1-2 can be combined arbitrarily, and the combined technical solutions all belong to the protection scope of the present application. The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. Use of a PPAR γ activator in the manufacture of a composition for improving the ability of an aquatic animal to resist environmental stress, wherein the composition comprises a PPAR γ activator.

2. The use of claim 1, wherein the improvement of the ability of the aquatic animal to resist environmental stress comprises one or more of promoting differentiation of adipocytes, reducing free fatty acid levels, reducing oxidative stress levels, improving resistance to cold stress, improving resistance to heat stress, and improving resistance to ammonia nitrogen stress.

3. A composition that can be administered to an aquatic animal, wherein the composition comprises a PPAR γ activator.

4. The composition according to claim 3, comprising 10-30mg of PPAR γ activator per kg of the composition.

5. The composition of claim 3, further comprising a pharmaceutically or feedstuffs or animal health acceptable carrier or excipient.

6. The composition of claim 3, wherein the composition is a pharmaceutical composition or an aquaculture feed additive or an aquatic animal health product.

7. The composition of claim 3, wherein the PPAR γ activator comprises one or more of rosiglitazone, ciglitazone, bezafibrate, and clofibrate.

8. A construction method of a zebra fish model for improving the environmental stress resistance of aquatic animals is characterized by comprising the following steps:

s1, knocking out PPAR gamma genes of the zebra fish to obtain mutant zebra fish;

s3, taking wild zebra fish as a wild control group and a wild experimental group respectively, and taking mutant zebra fish as a mutant experimental group, wherein the wild experimental group and the mutant experimental group are both fed with a composition added with a PPAR gamma activator;

9. The method of claim 8, wherein the detection of the indicators of the tissue sample comprises: one or more of liver triglyceride, liver free fatty acid, etc.

10. The method of claim 8, wherein the measure of resistance under stress comprises: survival rate under cold stress, survival rate under heat stress and survival rate under ammonia nitrogen stress.