Construction method and application of multi-organ development injury animal model based on non-coding long-chain RNA

Abstract

The invention discloses a construction method and application of a multi-organ development injury animal model based on non-coding long-chain RNA (Ribonucleic Acid). According to the present invention, it is found that the expression level of LncRNA URS0000A7E830 is significantly increased compared to the normal control group in the embryonic period amoxicillin exposed zebra fish model, and the URS0000A7E830 siRNA microinjection intervention is performed on the fertilized one-cell period zebra fish embryo based on the embryonic period amoxicillin exposed zebra fish model so as to effectively improve the multi-organ differentiation damage. The model established by the invention is novel, reliable, simple and convenient, and has important significance for illuminating the occurrence mechanism of the development injury of multiple organs and determining clinical early warning and intervention targets.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, specifically to a method for constructing and applying an animal model of multi-organ developmental injury based on non-coding long RNA. Background Technology

[0002] New types of toxic and harmful chemicals, or "new pollutants," have received widespread attention over the past decade. [1] Emerging pollutants include compounds generated by the adoption and application of new technologies, as well as compounds previously present in the environment but unidentifiable due to a lack of sensitive analytical methods and quantitative equipment. These emerging pollutants include antibiotics, antidepressants, hormones, nonsteroidal anti-inflammatory drugs (NSAIDs), food additives, and other chemicals. These substances can persist in the environment at trace concentrations and may affect aquatic organisms and humans. With the development of the pharmaceutical industry and the widespread use of drugs, the release of these molecules into the environment is almost inevitable. Previous animal experiments in our laboratory have also found that exposure to various exogenous substances (amoxicillin, dexamethasone, and ethanol, etc.) during pregnancy can induce multiple organ dysplasia in offspring. [2-4] However, its occurrence mechanism and early prevention and control targets are still unclear.

[0003] Zebrafish, as an excellent model organism, shares up to 71% genome homology with the human genome, exhibiting a variety of physiological and pharmacological similarities. Particularly in terms of homology with potential disease-related genes, 82% of the 3,176 potentially disease-causing genes listed in the human online Mendelian genetics database are homologous to at least one zebrafish gene. [5] In some cases, zebrafish models are easier to use for modeling human diseases than mouse or rodent models. For example, zebrafish are sensitive to thalidomide and can exhibit characteristic limb deformities and defects in homologous organs that are only found in rabbits and primates, including humans. [6] Zebrafish have high reproductive efficiency, producing a large number of embryos (including those with targeted mutations), making them suitable for cost-effective genetic and pharmacological screening. The zebrafish model organism is widely used to assess toxicity; models for studying potential hepatotoxicity have been established based on zebrafish. [7] Neurotoxicity [8] and cardiotoxicity [9] The model is noteworthy. Some studies have shown that adult zebrafish and zebrafish embryos / juveniles exhibit different effects in toxicity tests, which may help this invention differentiate and assess the different effects of drugs on pregnant women and fetuses.

[0004] Since their discovery in the 1950s, long noncoding RNAs (lncRNAs) have had their sequences, structures, and functions gradually revealed with the continuous development of high-throughput technologies. LncRNAs interact with proteins and nucleic acids in the cell nucleus and cytoplasm that regulate gene expression, contributing to the establishment of robust, flexible, and specific transcriptional and post-transcriptional regulatory mechanisms. [10, 11] This invention has successfully established a stable animal (zebrafish) model of multi-organ developmental injury for the first time. Through forward and reverse experiments, it was confirmed that LncRNA URS0000A7E830 can serve as an early screening and intervention target for multi-organ developmental injury, which is of great significance for screening the multi-organ developmental toxicity of environmental pollutants (including drugs).

[0005] References: 1.Bilal, M., et al., Emerging contaminants of high concern and theirenzyme-assisted biodegradation – A review. Environment International, 2019.124: p. 336-353. 2.Chen, Y., et al., Effects of amoxicillin exposure at different stages, doses and courses of pregnancy on adrenal development in fetal mice. Food Chem Toxicol, 2023. 175: p. 113754. 3.Liu, Y., et al., Developmental toxicity window of fetal testicularinjury in offspring mice induced by prenatal amoxicillin exposure at different time, doses and courses. Toxicol Lett, 2023. 374: p. 85-95. 4.Qin, J., et al., Impact of prenatal amoxicillin exposure onhippocampal development deficiency. Neuropharmacology, 2023. 223: p. 109331. 5.Howe, K., et al., The zebrafish reference genome sequence and itsrelationship to the human genome. Nature, 2013. 496(7446): p. 498-503. 6.Dong, W., et al., Augmentation of Pectoral Fin Teratogenicity byThalidomide in Human Cytochrome P450 3A-Expressing Zebrafish. Pharmaceuticals(Basel), 2023. 16(3). 7.Shihana, F., et al., Investigating the role of lipid genes in liverdisease using fatty liver models of alcohol and high fat in zebrafish (Daniorerio). Liver Int, 2023. 43(11): p. 2455-2468. 8.Mahmood, F., et al., A zebrafish model of CLN2 disease is deficientin tripeptidyl peptidase 1 and displays progressive neurodegenerationaccompanied by a reduction in proliferation. Brain, 2013. 136(Pt 5): p. 1488-507. 9.Staudt, D. and D. Stainier, Uncovering the molecular and cellular mechanisms of heart development using the zebrafish. Annu Rev Genet, 2012.46: p. 397-418. 10.Herman, AB, D. Tsitsipatis, and M. Gorospe, Integrated lncRNA function upon genomic and epigenomic regulation. Mol Cell, 2022. 82(12): p.2252-2266. 11.Wachsmuth, M., M. Caudron-Herger, and K. Rippe, Genomeorganization: balancing stability and plasticity. Biochim Biophys Acta, 2008.1783(11): p. 2061-79. Summary of the Invention In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method and application for constructing a multi-organ developmental injury animal model based on non-coding long RNA that is highly successful, effective, reliable, reproducible, simple and easy to implement.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for constructing an animal model of multi-organ developmental injury based on LncRNAURS0000A7E830.

[0007] This invention discloses a method for constructing an animal model of multi-organ developmental injury based on LncRNAURS0000A7E830. This method achieves efficient construction of an animal model of multi-organ developmental injury by precisely regulating the expression of LncRNAURS0000A7E830 and combining it with the embryonic development characteristics of zebrafish. The specific steps are as follows: Step S1: Preparation of transgenic vector Using the pDB739Tol2 vector as the transgenic vector, linearization and dephosphorylation were performed to obtain a linearized plasmid. The pDB739Tol2 vector contains Tol2, URS0000A7E830, GFP, polyA, and Tol elements. The specific method for obtaining it is as follows: the 4×KGFP vector is double-digested with HindIII and BshT1 enzymes, and the full sequence of URS0000A7E830 is inserted. After confirmation that the sequence is correct, the pDB739Tol2 vector is obtained.

[0008] Step S2: Embryo microinjection The Tol2 transposase mRNA was premixed with the linearized LncRNAURS0000A7E830 overexpression plasmid DNA to prepare an injection mixture; wherein the concentration of the linearized plasmid DNA in the mixture was 12.5~50 ng / μL and the concentration of Tol2 transposase mRNA was 100 ng / μL.

[0009] Healthy wild-type zebrafish males and females were selected for mating in the same tank. After fertilization, within 20 minutes, the above mixture was injected into each zebrafish embryo using microinjection. The injection volume was controlled at 1-3 nl / embryo to ensure that the exogenous gene was stably integrated into the zebrafish genome.

[0010] Step S3: Model Culture and Sample Collection The injected zebrafish embryos were placed in a suitable culture environment and cultured until 5 days post-fertilization (5dpf). The zebrafish larvae at this time were then collected, which constituted the completed animal model of multi-organ developmental injury, which can be used for subsequent detection of multi-organ developmental injury.

[0011] Among them, the relevant indicators for the detection of multi-organ developmental damage include, but are not limited to: light-dark alternation behavior test (used to assess neurodevelopment-related functions), gene detection of organ functions (such as the expression level detection of genes related to the functions of key organs such as heart, liver, and kidney), and morphological detection of organs (observing the morphological and structural changes of each organ through microscopic observation or tissue section staining).

[0012] Secondly, the present invention also provides two applications of the animal model constructed by the above method.

[0013] 1. Application of Animal Models in Drug Screening / Preparation: The multi-organ developmental injury animal models obtained by the above construction methods can be used to screen / prepare drugs for the prevention and control of environmental pollutants. Specifically, the candidate drugs to be screened are applied to the animal model, and the improvement effect of the candidate drugs on multi-organ developmental injury caused by environmental pollutants is evaluated by detecting the multi-organ developmental injury indicators of the model animals (such as the aforementioned light-dark alternation behavior, organ function genes, and organ morphological indicators). This allows for the screening of drugs with the function of preventing and controlling environmental pollutants, or the optimization of drug preparation processes based on the model.

[0014] 2. The animal model of multi-organ developmental injury obtained by the above construction method can also be used to screen / prepare drugs for the prevention and treatment of multi-organ developmental injury. Using this model as a disease model of multi-organ developmental injury, the repair or alleviation effects of candidate drugs on multi-organ developmental injury in model animals can be tested. Based on the test results, the effectiveness and appropriate dosage of the drugs can be determined, providing a reliable in vivo experimental basis for the development of drugs for the treatment of multi-organ developmental injury.

[0015] Thirdly, the present invention also provides an application of LncRNAURS0000A7E830 as a toxic target.

[0016] LncRNAURS0000A7E830 can serve as a toxicity target for screening / preparing drugs to prevent and treat environmental pollutants with multi-organ developmental damage effects. By monitoring the regulatory effect of candidate drugs on LncRNAURS0000A7E830 expression levels, combined with changes in multi-organ developmental damage indicators, it can be determined whether drugs can inhibit or alleviate multi-organ developmental damage induced by environmental pollutants by acting on this target, providing new targets and technical ideas for the development of targeted prevention and treatment drugs.

[0017] The technical principles and research process of this invention are as follows: This invention constructs a zebrafish model of multi-organ developmental injury by microinjecting the lncRNA URS0000A7E830 overexpression plasmid into one-cell stage zebrafish embryos. The model was then analyzed by detecting genes involved in multi-organ differentiation. Sox2, Nr5a1 , Foxa3 Gata4, Hhex and Gata6 It was found that after overexpression of LncRNA URS0000A7E830, the expression of genes related to differentiation function in the brain, renal interstitium, liver, and heart was significantly reduced, and different degrees of developmental loss phenotypes appeared in each organ.

[0018] This invention also revealed that the expression level of LncRNA URS0000A7E830 was significantly increased in embryonic exogenous exposure (amoxicillin) compared to the normal control group. In zebrafish, the effects of amoxicillin exposure on the differentiation functions of the zebrafish brain, interrenal glands, liver, and heart were clarified; high concentrations of amoxicillin significantly promoted the expression of LncRNA URS0000A7E830. This suggests that screening for developmental toxicity targets using LncRNA URS0000A7E830 is feasible.

[0019] Furthermore, this invention, at the overall level, uses a zebrafish model of embryonic amoxicillin exposure to intervene with one-cell stage zebrafish embryos through microinjection of LncRNA URS0000A7E830 siRNA, and detects the expression of LncRNA URS0000A7E830 in juvenile fish at 5 days post-fertilization (dpf). The results showed that inhibiting the expression of LncRNA URS0000A7E830 effectively alleviated the low differentiation function of multiple organs in zebrafish caused by embryonic amoxicillin exposure.

[0020] The advantages and beneficial effects of this invention are as follows: This model simulates the disease phenotype of embryonic multi-organ developmental damage in zebrafish, which is of great significance for elucidating the occurrence and development mechanism of multi-organ developmental loss and for determining early warning and prevention. Specific advantages are as follows: 1. The model construction of this invention is precise and efficient. It is constructed using a specific pDB739Tol2 vector and the injection time (within 20 minutes after fertilization), dosage (1~3 nl / particle), and concentration (plasmid DNA 12.5~50 ng / μL, transposase mRNA 100 ng / μL) are strictly controlled to ensure stable integration of the exogenous gene. 2. The model construction cycle of this invention is short; the model can be obtained after culturing to 5 dpf, resulting in a short cycle. 3. The detection system of this invention is comprehensive, covering multiple dimensions of indicators such as light-dark alternation behavior, organ function genes, and organ morphology. It can accurately assess the developmental damage of multiple organs, and the indicators selected in this model have good stability and strong repeatability. 4. This invention has a clearly defined target, with LncRNA URS0000A7E830 serving as the core regulatory target, providing a reliable method for constructing embryonic multi-organ developmental injury and offering a clear direction for studying the mechanisms of multi-organ developmental injury; 5. The zebrafish animal model of multi-organ developmental injury constructed based on this invention can be used to screen environmental pollutants (including drugs) for the treatment of multi-organ developmental injury, providing a reliable in vivo experimental platform for the screening and preparation of drugs for the prevention and treatment of environmental pollutants and drugs for the treatment of multi-organ developmental injury, and helping to efficiently develop targeted drugs; LncRNA URS0000A7E830, as a toxicity target, provides a new approach for developing targeted drugs to prevent and treat multi-organ developmental injury caused by environmental pollutants, filling the gap in targeted research in related fields, and is of great significance for protecting biological health and promoting pharmaceutical research and development. Attached Figure Description

[0021] Figure 1 Overexpression of LncRNA URS0000A7E830 leads to multi-organ developmental damage in zebrafish; Figure 1 A: Flowchart of zebrafish model construction for multiple organ injury; B: Plasmid map of URS0000A7E830; C: Morphology (20×) of 5 dpf juvenile fish microinjected with 25, 50, and 100 ng / μL overexpression plasmids; D: Synaptic protein I overexpressed in 5 dpf juvenile fish. syn1 mRNA expression level; E: 5 dpf overexpression of juvenile SRY-related HMG-box transcription factor 2 ( sox2 mRNA expression levels; F: Representative movement trajectories and heatmaps of 5 dpf zebrafish juveniles during three light-dark cycles; G: 5 dpf overexpression of juvenile sterol-1 ( nr5a1a mRNA expression level; H: 5 dpf overexpression of acute regulatory protein for sterol synthesis in juvenile fish ( star mRNA expression level; I: 5 dpf overexpression of juvenile fish cytochrome P450 17A1 ( cyp17a1 mRNA expression level; J: 5dpf overexpression of juvenile harpoon head box protein A3 ( foxa3 mRNA expression level; K: 5 dpf overexpression of juvenile fish GATA-binding protein 6 ( gata6 mRNA expression level; L: 5 dpf overexpression of juvenile fish fatty acid synthase ( fasn mRNA expression level; M: 5 dpf overexpression of juvenile fish 3-hydroxy-3-methylglutaryl-CoA reductase ( hmgcr mRNA expression level; N: 5 dpf overexpression of recombinant human homeobox protein in juvenile fish ( hhex mRNA expression level.

[0022] Figure 2 Amoxicillin exposure during the embryonic period can cause changes in the overall morphology of zebrafish; Figure 2A: Overall morphology of zebrafish fry 3, 4, and 5 days after fertilization; B: Changes in body length of zebrafish fry 3, 4, and 5 days after fertilization; C: Changes in head length of zebrafish fry 3, 4, and 5 days after fertilization.

[0023] Figure 3 Amoxicillin exposure during the embryonic period can cause damage to the differentiation function of multiple organs in zebrafish; Figure 3 Chinese: A: 5 dpf amoxicillin exposure juvenile fish synaptic protein I ( syn1 mRNA expression level; B: 5 dpf amoxicillin-exposed juvenile fish SRY-associated HMG-box transcription factor 2 ( sox2 C: Behavioral movement patterns and heatmaps of 5 dpf zebrafish juveniles during three light and dark cycles (n=6); D: Steroid factor-1 in 5 dpf zebrafish juveniles exposed to amoxicillin ( nr5a1a mRNA expression level; E: 5 dpf amoxicillin-exposed juvenile fish cytochrome P450 17A1 ( cyp17a1 mRNA expression level; F: 5 dpf amoxicillin exposure in juvenile fish acute regulatory protein of sterol synthesis ( star mRNA expression level; G: 5dpf zebrafish interrenal gland nr5a1 In situ hybridization (20×); H: 5 dpf amoxicillin exposed ventricular myosin heavy chain ( vmhc mRNA expression level; I: 5 dpf amoxicillin-exposed atrial myosin heavy chain ( amhc mRNA expression level; J: 5 dpf amoxicillin-exposed juvenile fish GATA-binding protein 4 ( gata4 mRNA expression level; K: 5 dpf zebrafish Tg (vMHC:ala) juvenile atrial morphology; L: 5 dpf amoxicillin-exposed juvenile forkhead box protein A3 ( foxa3 mRNA expression level; M: 5 dpf amoxicillin-exposed juvenile fish GATA-binding protein 6 ( gata6 mRNA expression level; N: 5 dpf overexpression of recombinant human homeobox protein in juvenile fish ( hhex mRNA expression level; O: 5 dpf overexpression of juvenile fish 3-hydroxy-3-methylglutaryl-CoA reductase ( hmgcr mRNA expression level; P: 5 dpf overexpression of fatty acid synthase in juvenile fish ( fasn Q: mRNA expression level; R: morphology of liver in 5 dpf zebrafish Tg (fabp10a:mCherry; ela:eGFP) juveniles; R: electron micrograph of local mitochondria in the liver of 5 dpf zebrafish.

[0024] Figure 4Intervention with lncRNA URS0000A7E830 siRNA can alleviate amoxicillin-induced multi-organ differentiation damage during the embryonic period; Figure 4 A: Somatic morphology of control, amoxicillin exposure, and lncRNA / siRNA microinjection treatment (20×) at 5 dpf; B: Body length of zebrafish after 5 dpf lncRNA / siRNA microinjection; C: mRNA expression of zebrafish URS0000A7E830 at 5 dpf; D: Knockdown of synaptic protein I in juvenile fish at 5 dpf. syn1 mRNA expression level; E: 5 dpf knockdown of juvenile fish SRY-related HMG-box transcription factor 2 ( sox2 ) mRNA expression level; F: Behavioral movement trajectory and heatmap of zebrafish juveniles in 3 light and dark cycles after 5 dpf siRNA intervention (n=8); G: 5 dpf knockdown of sterol-1 in juvenile fish ( nr5a1a mRNA expression level; H: 5 dpf knockdown of acute regulatory protein of sterol synthesis in juvenile fish ( star mRNA expression level; I: 5 dpf knockdown of juvenile fish cytochrome P450 17A1 ( cyp17a1 mRNA expression level; J: 5 dpf knockdown of juvenile fish 3-hydroxy-3-methylglutaryl-CoA reductase ( hmgcr mRNA expression level; K: 5 dpf knockdown of juvenile harpoon head box protein A3 ( foxa3 mRNA expression level; L: 5 dpf knockdown of fatty acid synthase in juvenile fish ( fasn mRNA expression level; M: 5 dpf knockdown of recombinant human homeobox protein in juvenile fish ( hhex mRNA expression level. Detailed Implementation

[0025] The technical content of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0026] The reagents used in the experiments of this invention were purchased from domestic or international markets, or prepared by the user according to the formula in the instruction manual; the experimental methods not specifically described are all conventional methods known in the art.

[0027] Example 1: Construction of the zebrafish model of embryonic multi-organ developmental injury according to the present invention 1. Laboratory animals Zebrafish were adapted to light / dark cycles in a constant-temperature (27±0.5°C) dechlorinated tap water recirculation system, with a 14h / 10h cycle. All experiments were conducted in accordance with institutional and national animal welfare guidelines and approved by the Institutional Animal Care and Use Committee of the Animal Experiment Center, Wuhan University. The zebrafish strains used in this study included the wild-type AB strain and the transgenic strains Tg(vmhc:mCherry) and Tg(fabp10a:mCherry; ela:eGFP).

[0028] 2. Experimental Methods Male and female AB strains and transgenic zebrafish were placed in mating tanks at a 1:2 ratio at 7 PM the day before model construction to acclimatize. The partition in the mating tanks was removed between 8 and 9 AM the following morning, and fertilized eggs were collected from the bottom of the tanks every 15 minutes. The fertilized eggs were placed in 90 mm culture dishes, and a mixture of 12.5–50 ng / μL URS0000A7E830 overexpression plasmid and 100 ng / μL transposase mRNA was prepared. 1–3 nmol of this mixture was injected into each one-cell stage embryo via microinjection.

[0029] Add 30ml of dechlorinated tap water. During the first 0-5 days after fertilization, replace the culture water with an equal volume of 15ml every 12 hours. Remove any dead embryos.

[0030] 3. Plasmid construction sequence The linearized full-length sequence of the plasmid is shown in SEQ ID NO.29.

[0031] 4. Detection Indicators and Methods 4.1 Real-time quantitative PCR experiment RNA was extracted from juvenile fish using Trizol reagent and then reverse transcribed to obtain cDNA. The cDNA, upstream and downstream primers for each gene, and SYBR Green I fluorescent dye were mixed thoroughly and then placed in an RT-qPCR instrument. The RT-qPCR reaction conditions were as follows (reaction volume: 10 µL): 95℃ pre-denaturation for 2 min; 95℃ denaturation for 10 s, 62℃ annealing for 30 s; 72℃ extension for 15 s, for a total of 45 cycles. Cox4i was used as an internal control. -△△Ct The relative expression levels of the target gene mRNA were calculated. Primer sequences are shown in Table 1 below: Table 1. Primer sequences

[0032] 4.2 Multiple organ morphological examination Before overall bright-field imaging, zebrafish juveniles at 3, 4, and 5 days post-fertilization were placed in 90 mm culture dishes (6 fish per group). After anesthetizing the juveniles with 1 mg / mL tricaine (ethyl 3-aminobenzoate, Sigma) in the culture water, they were observed under a microscope. Images were taken using an Olympus AH-2 optical microscope (Tokyo, Japan). Phenotypic quantification of body length and head length was performed using ImageJ 1.53c software.

[0033] Zebrafish larvae of the liver-fluorescent strain Tg (fabp10a: mCherry; ela: eGFP) at days 3, 4, and 5 post-fertilization were anesthetized with 1 mg / mL tricaine (ethyl 3-aminobenzoate, Sigma), and then fixed in 1% low-melting-point agarose gel with their left side facing upwards. Liver images were obtained using a Nikon SMZ25 fluorescence stereomicroscope.

[0034] Digoxigenin-labeled antisense RNA probes were used to investigate the renal interstitial glands. nr5a1 In situ hybridization was performed. Zebrafish embryos were fixed in 4% paraformaldehyde (PFA) and incubated overnight at 4°C. After washing with PBS, the embryos were digested with 20 µg / mL PBST (0.1% Tween 20) proteinase K and refixed with 4% PFA for 20 min. Pre-hybridization was performed at 68°C in hybridization buffer for 3–4 h, followed by overnight incubation at 68°C with diluted probe (1 ng / µL). Detection was performed using a 1:4000 dilution of anti-digoxigenin-AP antibody, and visualization was achieved via NBT / BCIP substrate reaction. To generate antisense ribose probes, the transcription template was amplified from cDNA and subcloned into the pGEM-TEasy vector (Vazyme Biotech Co., Ltd, Nanjing, China). The linearized construct was transcribed in vitro using T7 RNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) and a DIG RNA marker mixture (Roche, Basel, Switzerland).

[0035] 4.3 Behavioral testing of juvenile fish 5 days after fertilization Five-day-old zebrafish fry were prepared. Healthy fry at the same developmental stage were selected to ensure comparability of experimental results. The fry were placed in 24-well plates. An appropriate amount of culture medium was added to each well, with one fry per well. The plates were placed in a temperature-controlled incubator to maintain suitable environmental conditions. The camera and support were positioned to ensure clear image capture of the fry within the wells. Behavioral tracking software was used to connect a high-definition camera to capture the zebrafish's movement trajectories in real time, obtaining corresponding data and charts. The software was used to perform quantitative analysis on the fry, identifying and extracting relevant behavioral parameters.

[0036] 5. Experimental Results The effects of target lncRNAs on multi-organ differentiation function were verified by microinjecting them into zebrafish embryos during the embryonic stage. Figure 1 (A). This invention established microinjection groups of plasmids at different concentrations of 0, 12.5, 25, and 50 ng / L, and conducted intervention experiments in zebrafish embryonic organelles. The results showed that with increasing overexpression concentration, 5-day-fed juveniles developed spinal curvature deformities and systemic edema. Figure 1 In the middle B group, the mortality rate increased significantly, and the body length and head length decreased significantly. Furthermore, this invention examined changes in functional genes of various organs and found that the brain... Sox2 mRNA expression decreased significantly. Syn1 mRNA expression at 25 ng / The concentration of L decreased significantly ( P <0.05, Figure 1 (C). Meanwhile, behavioral results regarding the nervous system showed that in a dark environment, the lncRNA injection group at 25 ng / L activity decreased, at 50 ng / L showed a certain increase in central rest and activity disorder, with a significant increase in cumulative rest time. P <0.05, Figure 1 In the middle D group, decreased movement distance, increased resting frequency, and decreased angular velocity were observed. Under light stimulation, the lncRNA injection group showed increased activity, decreased cumulative resting time, increased movement distance, increased agitation frequency, and increased angular velocity with increasing concentration. (Adrenal glands) Nr5a1, Cyp17a1, StAR mRNA expression was significantly reduced ( P <0.05, Figure 1 (E), and there is a dose-effect relationship. Liver Fasn and Hmgcr mRNA expression decreased significantly at all concentrations. Hhex, Foxa3, Gata6 mRNA expression was higher than 25 ng / The concentration of L decreased significantly ( P <0.05, Figure 1 (F). The results above show that overexpression of URS0000A7E830 can induce a decline in the basic functions of multiple organs. Differentiation-related genes are more sensitive to overexpression. The sensitivity of each organ to lncRNA is ranked as follows: liver = renal gland > heart = brain.

[0037] Example 2: Screening for potential multi-organ developmental toxic drugs using the model of this invention 1. Laboratory animals: Zebrafish were adapted to light / dark cycles in a constant-temperature (27±0.5°C) dechlorinated tap water recirculation system, with a 14h / 10h cycle. All experiments were conducted in accordance with institutional and national animal welfare guidelines and approved by the Institutional Animal Care and Use Committee of the Animal Experiment Center, Wuhan University. The zebrafish strains used in this study included the wild-type AB strain and the transgenic strains Tg(vmhc:mCherry) and Tg(fabp10a:mCherry; ela:eGFP).

[0038] 2. Experimental methods: Male and female AB strains and transgenic zebrafish were placed in mating tanks at a 1:2 ratio at 7 PM the day before model construction to acclimatize. The partition in the mating tanks was removed between 8 and 9 AM the following morning, and fertilized eggs were collected from the bottom of the tanks 2 hours later. The fertilized eggs were placed in 90 mm culture dishes, and 10, 50, and 250 μM amoxicillin solutions were prepared using dechlorinated tap water. 30 mL of each solution was added to each solution to culture the embryos. During days 0-5 post-fertilization, the culture water was replaced every 12 hours with an equal volume of 15 mL. Dead embryos were removed.

[0039] 3. Detection indicators and methods 3.1 Real-time quantitative PCR experiment RNA was extracted from juvenile fish using Trizol reagent and then reverse transcribed to obtain cDNA. The cDNA, upstream and downstream primers for each gene, and SYBR Green I fluorescent dye were mixed thoroughly and then placed in an RT-qPCR instrument. The RT-qPCR reaction conditions were as follows (reaction volume: 10 µL): 95℃ pre-denaturation for 2 min; 95℃ denaturation for 10 s, 62℃ annealing for 30 s; 72℃ extension for 15 s, for a total of 45 cycles. Cox4i was used as an internal control. -△△Ct The relative expression level of the target gene mRNA was calculated. Primer sequences are shown in Table 1.

[0040] 3.2 Multiple organ morphological examination Before overall bright-field imaging, zebrafish juveniles at 3, 4, and 5 days post-fertilization were placed in 90 mm culture dishes (6 fish per group). After anesthetizing the juveniles with 1 mg / mL tricaine (ethyl 3-aminobenzoate, Sigma) in the culture water, they were observed under a microscope. Images were taken using an Olympus AH-2 optical microscope (Tokyo, Japan). Phenotypic quantification of body length and head length was performed using ImageJ 1.53c software.

[0041] Zebrafish larvae of the liver-fluorescent strain Tg (fabp10a:mCherry;ela:eGFP) at days 3, 4, and 5 post-fertilization were anesthetized with 1 mg / mL tricaine (ethyl 3-aminobenzoate, Sigma). The larvae were then fixed in 1% low-melting-point agarose gel with their left side facing upwards. Liver images were obtained using a Nikon SMZ25 fluorescence stereomicroscope.

[0042] Digoxigenin-labeled antisense RNA probes were used to investigate the renal interstitial glands. Nr5a1 In situ hybridization was performed. Zebrafish embryos were fixed in 4% paraformaldehyde (PFA) and incubated overnight at 4°C. After washing with PBS, the embryos were digested with 20 µg / mL PBST (0.1% Tween 20) proteinase K and refixed with 4% PFA for 20 min. Pre-hybridization was performed at 68°C in hybridization buffer for 3–4 h, followed by overnight incubation at 68°C with diluted probe (1 ng / µL). Detection was performed using a 1:4000 dilution of anti-digoxigenin-AP antibody, and visualization was achieved via NBT / BCIP substrate reaction. To generate antisense ribose probes, the transcription template was amplified from cDNA and subcloned into the pGEM-TEasy vector (Vazyme Biotech Co., Ltd, Nanjing, China). The linearized construct was transcribed in vitro using T7 RNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) and a DIG RNA marker mixture (Roche, Basel, Switzerland).

[0043] 3.3 Behavioral testing of juvenile fish 5 days after fertilization Five-day-old zebrafish fry were prepared. Healthy fry at the same developmental stage were selected to ensure comparability of experimental results. The fry were placed in 24-well plates. An appropriate amount of culture medium was added to each well, with one fry per well. The plates were placed in a temperature-controlled incubator to maintain suitable environmental conditions. The camera and support were positioned to ensure clear image capture of the fry within the wells. Behavioral tracking software was used to connect to a high-definition camera to capture the zebrafish's movement trajectories in real time, obtaining corresponding data and charts. The software was used to perform quantitative analysis on the fry, identifying and extracting relevant behavioral parameters.

[0044] 4. Experimental Results This invention established concentration gradient groups of 10, 50, and 250 µM, as well as time gradient groups of 3, 4, and 5 days of exposure at 250 µM, to obtain zebrafish juvenile samples. The results showed that amoxicillin exposure led to increased spinal curvature and a significant reduction in overall body length / head length in zebrafish, with a clear dose- and time-dependent effect. Figure 2(A and B) In summary, amoxicillin exposure during the embryonic period can cause overall developmental damage in zebrafish larvae and has certain potential toxicity.

[0045] This invention observed morphological and functional gene alterations in the brain, interrenal glands, heart, and liver of zebrafish under amoxicillin exposure. RT-qPCR results revealed key genes in the brain that promote neural stem cell differentiation. Sox2 and synaptic protein genes Syn1 Expression was significantly reduced under amoxicillin exposure ( P <0.05, Figure 3 (A). To investigate the nervous system development of juvenile fish, behavioral tests were conducted on 5-day-pass (dpf) juvenile fish at concentrations of 125, 250, and 500 µM. The results showed that in the dark environment, the amoxicillin group exhibited increased disorder and randomness in trajectory, with a dose-response effect, and a significantly increased cumulative resting time. P <0.05, Figure 3 In the B and C groups, indicators such as movement distance, angular velocity, manic frequency, and active frequency also showed varying degrees of decrease. Simultaneously, the response to light stimulation weakened, and activity under strong light increased with increasing concentration. The cumulative resting time significantly decreased, and indicators such as movement distance, angular velocity, manic frequency, and active frequency all showed varying degrees of decrease. Furthermore, this invention targets the renal gland function gene. Nr5a1, Cyp17a1 StAR The tests were conducted, and the results showed that the renal interstitial differentiation index in the amoxicillin group was significantly higher than that in the control group. Nr5a1, Cyp17a1 Expression decreased in a dose-dependent manner. StAR Expression increased in a dose-dependent manner, and was significantly elevated at high concentrations. P <0.05, Figure 3 (D). Nr5a1 In situ hybridization results showed that amoxicillin exposure reduced the adrenal gland area in 5-day-fed zebrafish juveniles, and there was a certain dose-response effect. Figure 3 (E). Next, the invention tested cardiac function, and the results showed early myocardial differentiation markers. Gata4 Expression was significantly reduced, atrial marker genes showed no significant changes, while ventricular marker gene expression increased with increasing amoxicillin concentration. Figure 3 (middle F) amhc and vmhc The fluorescent reporter strain showed no significant changes in cardiac morphology and structure under amoxicillin exposure. Figure 3 (G). Finally, this invention detected functional genes related to liver differentiation and lipid metabolism, and the results showed that, Foxa3, Gata4, Hhex Expression was significantly reduced. Fasn, Hmgcr Significantly increased expression ( Figure 3 (in H), and in fabp10a A certain degree of liver developmental delay was observed in the liver fluorescent strain. Figure 3In the middle I), local mitochondrial hypertrophy and vacuolation can be observed in the liver under electron microscopy. Figure 3 (J). Finally, this invention detected the expression level of URS0000A7E830 in zebrafish juveniles at different concentrations of 5 dpf, and found that amoxicillin exposure significantly increased the expression of URS0000A7E830. Figure 3 (K). In summary, amoxicillin exposure can cause developmental damage to multiple organs, with a significant reduction in differentiation function, and this differentiation impairment is caused by an increase in URS0000A7E830.

[0046] Example 3: Study on the improvement of multi-organ developmental damage based on the intervention of the model target of the present invention 1. Laboratory animals: Zebrafish were adapted to light / dark cycles in a constant-temperature (27±0.5°C) dechlorinated tap water recirculation system, with a 14h / 10h cycle. All experiments were conducted in accordance with institutional and national animal welfare guidelines and approved by the Institutional Animal Care and Use Committee of the Animal Experiment Center, Wuhan University. The zebrafish strains used in this study included the wild-type AB strain and the transgenic strains Tg(vmhc:mCherry) and Tg(fabp10a:mCherry; ela:eGFP).

[0047] 2. Experimental methods: Male and female AB strains and transgenic zebrafish were placed in mating tanks at a 1:2 ratio after 7 PM the day before the experiment to acclimatize. The partition in the mating tanks was removed between 8 and 9 AM the following morning, and fertilized eggs were collected from the bottom of the tanks every 15 minutes. The fertilized eggs were placed in 90 mm culture dishes, and 1–3 mL of a 50 ng / μL URS0000A7E830 siRNA solution was prepared and injected into each one-cell stage embryo via microinjection.

[0048] Add 30 ml of 250 µM amoxicillin solution. During days 0-5 post-fertilization, replace the culture water with an equal volume of 15 ml every 12 hours. Remove any dead embryos.

[0049] 3. Detection indicators and methods 3.1 Real-time quantitative PCR experiment RNA was extracted from juvenile fish using Trizol reagent and then reverse transcribed to obtain cDNA. The cDNA, upstream and downstream primers for each gene, and SYBR Green I fluorescent dye were mixed thoroughly and then placed in an RT-qPCR instrument. The RT-qPCR reaction conditions were as follows (reaction volume: 10 µL): 95℃ pre-denaturation for 2 min; 95℃ denaturation for 10 s, 62℃ annealing for 30 s; 72℃ extension for 15 s, for a total of 45 cycles. Cox4i was used as an internal control.-△△Ct The relative expression level of the target gene mRNA was calculated. Primer sequences are shown in Table 1.

[0050] 3.2 Multiple organ morphological examination Before overall bright-field imaging, zebrafish juveniles at 3, 4, and 5 days post-fertilization were placed in 90 mm culture dishes (6 fish per group). After anesthetizing the juveniles with 1 mg / mL tricaine (ethyl 3-aminobenzoate, Sigma) in the culture water, they were observed under a microscope. Images were taken using an Olympus AH-2 optical microscope (Tokyo, Japan). Phenotypic quantification of body length and head length was performed using ImageJ 1.53c software.

[0051] 3.3 Behavioral testing of juvenile fish 5 days after fertilization Five-day-old zebrafish fry were prepared. Healthy fry at the same developmental stage were selected to ensure comparability of experimental results. The fry were placed in 24-well plates. An appropriate amount of culture medium was added to each well, with one fry per well. The plates were placed in a temperature-controlled incubator to maintain suitable environmental conditions. The camera and support were positioned to ensure clear image capture of the fry within the wells. Behavioral tracking software was used to connect a high-definition camera to capture the zebrafish's movement trajectories in real time, obtaining corresponding data and charts. The software was used to perform quantitative analysis on the fry, identifying and extracting relevant behavioral parameters.

[0052] 4. Experimental Results This invention involves microinjecting target lncRNA siRNA into zebrafish embryos during the embryonic stage, followed by exposure of the embryos to 250 µM amoxicillin for 5 days. Body length and gene expression in various organs of the juvenile fish were measured. Results showed that after siRNA silencing of lncRNA, spinal curvature disappeared in 5-day-old juveniles under bright field conditions. Figure 4 (A), and the height has significantly increased ( Figure 4 (B) RT-qPCR was used to detect the knockdown efficiency of siRNA against lncRNA, verifying the effectiveness of siRNA. Figure 4 (C). Furthermore, this invention detected changes in functional genes of various organs, including the adrenal glands. Cyp17a1 Significantly rebounded to normal levels. Nr5a1a There is an upward trend. StAR The expression levels were all significantly reduced to normal levels. P <0.05, Figure 4 (Middle D) Brain Sox2, Syn1, Snap25 mRNA expression increased significantly ( P <0.05, Figure 4 (E), behavioral results showed that the activity of juvenile fish tended to be normal under both dark and light conditions. Figure 4 (G) liver Fasn Significant pullback, HhexSignificant rebound, Hmgcr, Foxa3 The change in mRNA expression was not significant. Figure 4 (F). The above results show that silencing URS0000A7E830 can salvage the multi-organ function decline caused by amoxicillin.

Claims

1. A method for constructing a non-coding long-chain RNA-based multi-organ development impairment animal model, characterized by comprising the following steps: It comprises the following steps: ​ S1: linearization and dephosphorylation treatment is carried out by taking pDB739Tol2 vector as transgenic vector to obtain linearized plasmid, wherein the pDB739Tol2 vector comprises element Tol2-URS0000A7E830-GFP-polyA-Tol; S2: the Tol2 transposase mRNA is pre-mixed with LncRNA URS0000A7E830 overexpression plasmid DNA, and healthy wild-type zebrafish male and female fish are selected for combined mating, and each embryo is injected with the mixed solution by microinjection after fertilization; S3: the zebrafish larvae are cultured until fertilization, and the larvae are collected for subsequent detection.

2. The construction method according to claim 1, characterized in that: in S2: the healthy wild-type zebrafish male and female fish are microinjected within 20 minutes after combined mating and fertilization; and each embryo is injected with the mixed solution by microinjection after fertilization, and the amount of the mixed solution is 1-3 nl; in S3: the zebrafish larvae are cultured until 5 days after fertilization and collected.

3. The method of construction of claim 2, wherein: In S1: the pDB739Tol2 vector is obtained by inserting the whole sequence of URS0000A7E830 into the 4×KGFP vector after enzyme digestion by HindIII and BshT1.

4. The method of construction of claim 3, wherein: In the mixed solution of S2: the concentration of linearized plasmid DNA is 12.5-50 ng / μL, and the concentration of transposase mRNA is 25-100 ng / μL.

5. The method of construction of claim 3, wherein: In S3, the multi-organ development damage detection related indexes are: light-dark alternation behavior, organ function gene detection, and organ morphology detection.

6. The animal model constructed by the method of any one of claims 1-5 is applied to screening / preparing drugs for preventing and treating environmental pollutants.

7. The animal model constructed by the method of any one of claims 1-5 is applied to screening / preparing drugs for preventing and treating multi-organ development damage.

8. LncRNA URS0000A7E830 is applied to screening / preparing drugs for preventing and treating environmental pollutants with multi-organ development damage.

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