LncLRFIF and application thereof in preparation of medicine for treating fatty liver hemorrhage syndrome of laying hens

Abstract

The present application relates to the field of biological medicine, and particularly relates to lncLRFIF and application thereof in preparation of a medicine for treating laying hen fatty liver hemorrhagic syndrome.The present application provides lncLRFIF, and it is found for the first time that lncLRFIF can be used for preparation of a medicine for treating laying hen fatty liver hemorrhagic syndrome.In specific embodiments of the present application, lncLRFIF is knocked down by siRNA, so that the activities of ALT and AST in supernatant of chicken primary hepatocytes under FFAs treatment are recovered, lipid droplet accumulation and TG and T-CHO contents are significantly reduced, lipid synthesis is increased, lipid transport is increased, mitochondrial ATP and ROS contents are increased, and mitochondrial respiration and function are recovered.It can be seen that lncLRFIF can improve liver cell damage caused by FFAs and regulate lipid metabolism.The present application enriches the pathogenesis of laying hen fatty liver hemorrhagic syndrome, and provides a new idea for improving poultry egg production performance.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to an lncLRFIF and its application in the preparation of a drug for treating fatty liver hemorrhage syndrome in laying hens. Background Technology

[0002] Fatty liver hemorrhage syndrome (FLHS) is a common nutritional and metabolic disease in high-producing laying hens. It causes a sharp decline in egg production and, in extreme cases, sudden death, posing a serious threat to the development of the poultry industry. Due to the long and slow course of FLHS, numerous atypical and subclinical cases exist in laying hen production, severely restricting egg production performance and causing significant economic losses to my country's poultry farming industry.

[0003] Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs longer than 200 nucleotides. They play important roles in various biological processes, including gene expression regulation, cell differentiation, proliferation, and apoptosis. They can regulate gene expression, cell signaling, and metabolic pathways, thereby affecting cellular physiological functions and pathological processes. Most lncRNAs are transcribed by RNA polymerase II and carry a 5' cap and a 3' poly(A) tail. Some lncRNAs lack poly(A) modification due to alternative splicing. This characteristic leads to complex mechanisms that are not yet fully understood.

[0004] To date, relevant studies have shown that lncRNAs participate in the regulation of lipid metabolism through multiple mechanisms and play an important role in maintaining lipid metabolism balance and normal physiological functions, such as lncRNAs Snhg9, lncRHL, and long non-coding RNA-Hilnc. However, there are no reports on the regulatory and diagnostic effects of non-coding long RNAs on FLHS disease. Summary of the Invention

[0005] The purpose of this invention is to provide an lncLRFIF and its application in the preparation of a drug for treating fatty liver hemorrhage syndrome in laying hens, thereby addressing the problems existing in the prior art. This invention lays the foundation for developing nutritional intervention or gene therapy strategies targeting lncRNAs, and provides new ideas for alleviating the huge economic losses caused by metabolic diseases in the laying hen industry.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides an lncLRFIF (originally named ENSGALT00000095928), the nucleotide sequence of which is shown in SEQ ID NO.47.

[0008] This invention provides the use of the above-mentioned lncLRFIF in the preparation of medicaments for the prevention and / or treatment of fatty liver hemorrhage syndrome in laying hens.

[0009] Preferably, the drug achieves the effect of preventing and / or treating fatty liver hemorrhage syndrome in laying hens by increasing ALT and AST activity, reducing lipid droplet accumulation, reducing TG and T-CHO content, reducing lipid synthesis protein expression, increasing lipid transport protein expression, increasing ATP and ROS content, and increasing the expression of mitochondrial function-related proteins and mitochondrial respiratory chain-related proteins.

[0010] The lipid synthesis proteins include ACC1 protein, SREBP1 protein, and PPARγ protein.

[0011] The lipid transporter includes the APOA1 protein.

[0012] This invention provides a drug for the prevention and / or treatment of fatty liver hemorrhage syndrome in laying hens, the drug comprising an agent that inhibits the expression of the aforementioned lncLRFIF.

[0013] Preferably, the formulation comprises Gallus-silncLRFIF1; the nucleotide sequence of the sense strand of Gallus-silncLRFIF1 is shown in SEQ ID NO.41, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.42.

[0014] Preferably, the drug achieves the effect of preventing and / or treating fatty liver hemorrhage syndrome in laying hens by increasing ALT and AST activity, reducing lipid droplet accumulation, reducing TG and T-CHO content, reducing lipid synthesis protein expression, increasing lipid transport protein expression, increasing ATP and ROS content, and increasing the expression of mitochondrial function-related proteins and mitochondrial respiratory chain-related proteins.

[0015] The lipid synthesis proteins include ACC1 protein, SREBP1 protein, and PPARγ protein.

[0016] The lipid transporter includes the APOA1 protein.

[0017] Preferably, the drug also includes pharmaceutically acceptable excipients.

[0018] This invention provides the application of the above-mentioned lncLRFIF as a target in screening drugs for the prevention and / or treatment of fatty liver hemorrhage syndrome in laying hens.

[0019] This invention provides a method for screening drugs to treat fatty liver hemorrhage syndrome in laying hens, including the step of detecting the expression level of chicken lncLRFIF before and after drug administration; the nucleotide sequence of the lncLRFIF is shown in SEQ ID NO.47.

[0020] This invention provides the application of the above-mentioned lncLRFIF in regulating lipid metabolism in laying hen liver.

[0021] Further optimization involves inhibiting the expression level of lncLRFIF to reduce the expression level of lipid synthesis proteins and increase the expression level of lipid transport proteins.

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

[0023] This invention provides an inncLRFIF, and for the first time, it has been discovered that inncLRFIF can be used to prepare a drug for treating fatty liver hemorrhage syndrome in laying hens. Pearson correlation analysis in specific embodiments of this invention shows that the inncLRFIF provided by this invention is significantly positively correlated with the comprehensive score of fatty liver in laying hens, body weight, blood glucose, free fatty acids, and aspartate aminotransferase, indicating that inncLRFIF can act as a key regulator of FLHS. Furthermore, after siRNA knockdown of inncLRFIF, the activities of ALT and AST in the supernatant of primary hepatocytes treated with FFAs were restored, lipid droplet accumulation was significantly reduced, TG and T-CHO contents were significantly decreased, the expression of lipid synthesis proteins was decreased, the expression of lipid transport proteins was increased, mitochondrial ATP and ROS contents were increased, and the expression of mitochondrial function-related proteins and mitochondrial respiratory chain-related proteins were increased, further demonstrating that inncLRFIF can improve hepatocyte damage caused by FFAs and regulate lipid metabolism. The results of specific embodiments of this invention show that lncLRFIF plays an important regulatory role in lipid metabolism in chicken primary hepatocytes. Therefore, the lncLRFIF provided by this invention can be used as a target for screening drugs to prevent and / or treat fatty liver hemorrhage syndrome in laying hens. Inhibitors of lncLRFIF can be used to prepare drugs to prevent and / or treat fatty liver hemorrhage syndrome in laying hens. This invention lays the foundation for further research on the mechanism of fatty liver hemorrhage syndrome in laying hens and provides new ideas for improving poultry egg production performance. Attached Figure Description

[0024] Figure 1The results of identification, localization, and analysis of lncLRFIF are shown below. A represents the lncLRFIF mRNA expression level in the liver tissue of laying hens; Control represents normal laying hens; FLHS represents laying hens with FLHS model fatty liver hemorrhage syndrome. B and C represent the subcellular localization analysis of lncLRFIF; Control represents untreated primary chicken hepatocytes; FFAs represent primary chicken hepatocytes treated with FFAs for 36 hours; GAPDH is a cytoplasmic internal control. D is a gel image after separation of the nucleus and cytoplasm.

[0025] Figure 2 For lncLRFIF Pearson correlation analysis;

[0026] Figure 3 Selection of lncLRFIF knockdown sequence and transfection concentration;

[0027] Figure 4 To knock down the effect of lncLRFIF on the activity of ALT and AST in chicken primary hepatocytes treated with FFAs;

[0028] Figure 5 To knock down the effect of lncLRFIF on the TG and T-CHO content of chicken primary hepatocytes treated with FFAs;

[0029] Figure 6 Oil Red staining (left side staining image) and quantitative analysis (right side bar chart); where siNC represents primary chicken hepatocytes transfected with siNC for 8 hours, silncLRFIF represents primary chicken hepatocytes transfected with Gallus-silncLRFIF for 18 hours, FFAs represent primary chicken hepatocytes treated with FFAs for 36 hours, and FFAs+silncLRFIF represents primary chicken hepatocytes treated with FFAs for 36 hours and transfected with Gallus-silncLRFIF for 18 hours.

[0030] Figure 7 To investigate the effect of lncLRFIF on the expression levels of lipid metabolism-related proteins in chicken primary hepatocytes treated with FFAs; the left side is a Western blotting plot, and the right side is a statistical plot of lipid metabolism-related protein expression levels.

[0031] Figure 8 To knock down the effect of lncLRFIF on ATP content in chicken primary hepatocytes treated with FFAs;

[0032] Figure 9 ROS fluorescence staining of chicken primary hepatocytes mitochondria;

[0033] Figure 10Flow cytometry analysis of ROS in mitochondria of primary chicken hepatocytes; the left side shows the ROS flow cytometry analysis, and the right side shows the ROS percentage statistics.

[0034] Figure 11 Fluorescence staining of mitochondrial membrane potential in primary chicken hepatocytes;

[0035] Figure 12 Flow cytometry analysis of mitochondrial membrane potential in primary chicken hepatocytes; the left side shows the flow cytometry analysis of mitochondrial membrane potential in primary hepatocytes, and the right side shows the proportion of cells with low membrane potential.

[0036] Figure 13 To investigate the effect of lncLRFIF on the expression levels of mitochondrial function-related proteins in chicken primary hepatocytes treated with FFAs; the left side is a Western blotting plot, and the right side is a statistical plot of mitochondrial function-related protein expression levels.

[0037] Figure 14 To investigate the effect of lncLRFIF on the expression levels of mitochondrial respiratory chain-related proteins in chicken primary hepatocytes treated with FFAs; the left side shows the Western blotting plot, and the right side shows the statistical plot of mitochondrial respiratory chain-related protein expression levels.

[0038] Figure 15 The results are validations for differentially expressed lncRNAs; where Control represents normal laying hens and FLHS represents the FLHS model.

[0039] Figure 16 To validate the results of differentially expressed miRNAs; where Control represents normal laying hens and FLHS represents the FLHS model.

[0040] Figure 17 To validate the differentially expressed mRNA results, Control represents normal laying hens, and FLHS represents the FLHS model. Detailed Implementation

[0041] Example 1

[0042] I. Acquisition and Identification of lncLRFIF (original name ENSGALT00000095928)

[0043] A pathological model of fatty liver hemorrhage syndrome (FLHS) in laying hens was constructed using a high-energy, low-protein diet. The construction method of this model followed the method described in the literature "Study on the Influence of Betaine and Biotin on the Expression of Muscle A-I and B-100 in Fatty Liver Hemorrhage Syndrome in Laying Hens" (Zhou Yujing. Study on the Influence of Betaine and Biotin on the Expression of Muscle A-I and B-100 in Fatty Liver Hemorrhage Syndrome in Laying Hens [D]. Jiangxi Agricultural University, 2011.). Subsequently, the liver of the model was subjected to transcriptomic sequencing; normal laying hens were used as controls (Control group).

[0044] The accuracy of non-coding RNA expression patterns in whole transcriptome sequencing data was verified by qPCR experiments. The primers used for qPCR are shown in Table 1.

[0045] Table 1 Primer Table

[0046]

[0047]

[0048] II. In vitro efficacy experiment

[0049] 1. Experimental materials

[0050] Experimental animals: The fertilized chickens used in this invention underwent routine disinfection and treatment, and were then incubated in a rotating incubator until 12 embryonic stages, subsequently used for the extraction of primary chicken hepatocytes. The incubator temperature was 37℃, and the humidity was 65%-70%RH.

[0051] The main reagents and consumables are shown in Table 2.

[0052] Table 2 Main reagents and consumables used in the experiment

[0053]

[0054]

[0055] 2. Experimental Methods

[0056] 2.2 Nucleus-Cytoplasmic Separation

[0057] RNA was isolated and extracted from the nuclei and cytoplasm of laying hen liver tissue according to the instructions of the nucleus-cytoplasm separation kit. The specific steps are as follows:

[0058] (1) Weigh 0.1g of laying hen liver tissue and add 1mL of Lysis Buffer J, then homogenize in a low-temperature tissue homogenizer; (2) Centrifuge at 4℃ and 12000×g for 10min; transfer the supernatant (cytoplasmic RNA component) to an EP tube to precipitate the nuclear RNA component; (3) Cytoplasmic RNA extraction: ① Add 1mL of Buffer SK to the cytoplasmic RNA component in step (2) and vortex to mix; ② Add 1mL of anhydrous ethanol and vortex to mix; ③ Collect the liquid from step ② into a cytoplasmic RNA collection column and centrifuge at 4℃ and 3500×g for 1min; ④ Repeat step ③; ⑤ Discard the supernatant and store the cytoplasmic RNA collection column for later use; (4) Nuclear RNA extraction: ① Add 2mL of Buffer J to the cytoplasmic RNA component in step (2) SK, vortex to mix; ② Add 2 mL of anhydrous ethanol, vortex to mix; ③ Collect the liquid in ② into the nuclear RNA adsorption column, centrifuge at 4℃ and 3500×g for 1 min; ④ Repeat step ③; ⑤ Discard the supernatant, and store the nuclear RNA adsorption column for later use; (5) Add 400 μL WashSolution A to the adsorption columns in steps (3) and (4) respectively, centrifuge at 4℃ and 12000×g for 1 min; (6) Repeat step (5) three times; (7) Discard the supernatant, centrifuge at 4℃ and 14000×g for 1 min, and store the empty column after centrifugation; (8) Add 50 μL Elution Buffer E to the adsorption column, centrifuge at 4℃ and 14000×g for 1 min, and elute; (9) Repeat the eluent to the adsorption column for a second elution, and the eluent is cytoplasmic / nuclear RNA; (10) See step “2.3” for the subsequent RNA reverse transcription and fluorescence quantitative steps.

[0059] 2.3 Quantitative Real-Time PCR Reaction

[0060] Total RNA was extracted from primary chicken hepatocytes using Trizol lysis buffer according to the manufacturer's instructions. RNA concentration was measured using a UV spectrophotometer. The RNA was then reverse-transcribed into cDNA according to the reverse transcription kit instructions. Using chicken liver cDNA as a template, the reaction was performed in a standard PCR instrument following the Novizan High Fidelity PCR Mix instructions. The reverse transcription and real-time quantitative PCR reaction systems and conditions are shown in Tables 3-6.

[0061] Table 3 Reverse transcription reaction system

[0062]

[0063]

[0064] Note: Gently pipette 8-10 times until fully mixed, then briefly centrifuge to collect the mixture at the bottom of the tube.

[0065] Table 4 Reverse transcription reaction conditions

[0066] Reaction conditions reaction time 50℃ 15min 85℃ 5s

[0067] Table 5 Real-time quantitative PCR reaction system

[0068] Components volume 2×ChamQUniversalSYBRqPCRMasterMix 5μL Upstream primer (10 μM) 0.2μL Downstream primer (10 μM) 0.2μL TemplateDNA / cDNA 1μL <![CDATA[ddH2O]]> 3.6μL Total 10μL

[0069] Note: The nucleotide sequences of the upstream and downstream primers are shown in Table 1.

[0070] Table 6 Real-time quantitative PCR reaction conditions

[0071]

[0072] 2.4 Design and Synthesis of siRNA

[0073] A specific interfering fragment (silncLRFIF) was designed based on the full-length lncLRFIF sequence, and a negative control fragment (siNC) was synthesized simultaneously. All fragments were purchased from Shanghai Hanheng Biotechnology Co., Ltd. The primer sequences are shown in Table 7. The selected silncLRFIF sequence was Gallus-silncLRFIF1.

[0074] Table 7 siRNA sequences

[0075]

[0076] 2.5 Isolation, culture, processing and transfection of primary chicken embryo hepatocytes

[0077] 2.5.1 Isolation and Culture of Primary Chicken Embryo Hepatocytes

[0078] (1) All instruments required are sterilized and dried by high temperature and high pressure. Before starting, the sterile operating table is disinfected by ultraviolet ventilation for 30 minutes; (2) Take 12-day-old chicken embryos, disinfect the eggshell with 75% alcohol and place it on the sterile operating table. Use tweezers to break the eggshell above the air cell and tear open the shell membrane. Replace the tweezers and take out the chicken embryo and place it in 1×PBS pre-cooled at 4℃; (3) Wash off the blood on the surface of the chicken embryo and peel off the liver in a sterile petri dish and place it in a new petri dish containing 1×PBS; (4) Remove the fascia, gallbladder and other parts attached to the liver and place the cleaned liver in a new petri dish containing 1×PBS; (5) Use ophthalmic scissors to cut the liver into pieces <1mm. 3(6) Add an equal volume of 1% type IV collagenase to the liver tissue pellet, place the EP tube in a 37°C water bath for 15 min, and shake the EP tube continuously during digestion to ensure that the liver tissue pellet is fully in contact with the type IV collagenase. (7) After digestion, add an equal volume of 1% type IV collagenase to the stop culture medium (basal culture medium containing 10% fetal bovine serum), and blow evenly with a Pasteur pipette until the tissue pellet is no longer visible. (8) Filter the cell suspension through 200-mesh and 400-mesh cell sieves. (9) Collect the filtrate into a 10 mL centrifuge tube, centrifuge at 1200 rpm for 5 min, and discard the supernatant. (10) Wash the pelleted cells with basal culture medium, centrifuge again at 1100 rpm for 3 min, and discard the supernatant. (11) Resuspend the cells in an appropriate amount of basal culture medium and count the cells.

[0079] 2.5.2 Cell grouping treatment

[0080] The experiment induced steatosis in chicken hepatocytes using 1 mM FFAs (OA (oleic acid):PA (palmitic acid) = 2:1 (V:V)). Some experiments were divided into two groups: a Control group and an FFAs group. The Control group consisted of untreated primary chicken embryo hepatocytes, while the FFAs group consisted of primary chicken embryo hepatocytes treated with FFAs for 36 hours. Other experiments were divided into four groups: siNC group, silncLRFIF group, FFAs group, and FFAs+silncLRFIF group. siNC consisted of primary chicken hepatocytes transfected with siNC for 8 hours, silncLRFIF consisted of primary chicken hepatocytes transfected with Gallus-silncLRFIF for 18 hours, FFAs consisted of primary chicken hepatocytes treated with FFAs for 36 hours, and FFAs+silncLRFIF consisted of primary chicken hepatocytes treated with FFAs for 36 hours and transfected with Gallus-silncLRFIF for 18 hours.

[0081] 2.5.3 Cell transfection

[0082] (1) The cells were loaded with 5 × 10 5(1) Spread the cells at a density of 50%-70% in a 12-well plate and transfect them when the cells have grown and aggregated to 50%-70%; (2) Take a 1.5 mL centrifuge tube, add 100 μL of DMEM medium and 3 μL of LipoFiter 3.0, mix gently and let stand for 5 min; (3) Take another 1.5 mL centrifuge tube, add 100 μL of DMEM medium and 150 nM of interfering RNA and / or 1 μg of plasmid, mix gently and let stand for 5 min; (4) Mix the liquids from steps (2) and (3) and let stand at room temperature for 20 min; (5) Discard the medium in the wells, wash the cells twice with 1×PBS, add 800 μL of serum-free basal medium to each well, and then add the liquid from step (4) evenly to the wells and culture in a cell culture incubator for 6-8 h; (6) Discard the medium in the wells, wash the cells twice with 1×PBS, and perform subsequent treatments on the cells according to the experimental requirements.

[0083] 2.6 Assay of ALT and AST activities in cell supernatant

[0084] Chicken primary hepatocytes were cultured at 5 × 10⁻⁶ 5 Cells were seeded at a density in 12-well plates. When the cells reached 80% confluence, they were treated with FFAs. After 36 hours of FFA treatment, the culture medium in the wells was collected for analysis. Following the instructions of the Nanjing Jiancheng reagent kit, the activities of ALT and AST in the cell supernatant were measured.

[0085] 2.7 Determination of cellular TG and T-CHO content

[0086] Chicken primary hepatocytes were cultured at 5 × 10⁻⁶ 5 Cells were seeded at a density in 12-well plates. When the cells reached 80% confluence, they were treated with FFAs. After 36 hours of FFA treatment, the culture medium in the wells was removed, and the cells were washed twice with 1×PBS. A suitable amount of trypsin was added for digestion, and the cells were collected. 1 mL of physiological saline was added to the cells, and the cells were sonicated (disruption conditions: ice bath, 200W power, 5 seconds run, 15 seconds interval, repeated 5 times) to prepare a cell homogenate. Following the instructions of the Nanjing Jiancheng reagent kit, the TG and T-CHO content of the cells was measured.

[0087] 2.8. Cell Oil Red Staining and Quantitative Analysis

[0088] Staining was performed using the Solarbio Oil Red O staining kit (cell-specific). Oil Red stained images were analyzed using ImageJ software; the area of ​​the red lipid droplets was collected, and the lipid droplet area ratio was obtained for data analysis.

[0089] 2.9. Cell Transmission Electron Microscopy Observation

[0090] Chicken primary hepatocytes were used at a concentration of 1×10⁻⁶ 6The cells were seeded at a density in 6-well plates. When the cells reached 80% confluence, they were treated with FFAs. After 36 hours of FFA treatment, the culture medium in the wells was removed, the cells were washed twice with 1×PBS, and the cells were digested with an appropriate amount of trypsin and the cell pellet was collected. The cell pellet was then fixed with 1 mL of electron microscopy fixative. Tissue was washed 2-3 times with PBS, and the surface liquid was blotted dry with sterile filter paper. The tissue blocks were then fixed in 1% osmium tetroxide solution at room temperature for 2 hours. Tissue was washed 2-3 times with PBS, and the surface liquid was blotted dry with sterile filter paper. The fixed samples were dehydrated with 30%, 50%, 70%, 80%, 90%, and 100% ethanol for 20 minutes each time, followed by dehydration with acetone twice for 15 minutes each time. The dehydrated samples were then gradually immersed in mixed solutions of acetone:epoxy resin 812 embedding agent = 2:1 (V:V), acetone:epoxy resin 812 embedding agent = 1:1 (V:V), and acetone:epoxy resin 812 embedding agent = 1:2 (V:V) for 4 hours, 4 hours, and 24 hours, respectively. The immersed samples were placed in embedding molds, pure embedding agent was added, and polymerization was carried out at 70°C. After the samples were removed and placed at room temperature, the tissue was positioned and trimmed. After repair, the sections were cut into 60-80 nm thin slices using an ultramicrotome for subsequent staining. The slices were immersed in 2% saturated uranium acetate staining solution and stained in the dark for 30 minutes, then rinsed thoroughly three times with double-distilled water. The water on the copper mesh was then blotted dry with sterile filter paper. The slices were then immersed in lead citrate staining solution and stained for 10 minutes, then rinsed thoroughly three times with double-distilled water. The water on the copper mesh was then blotted dry with sterile filter paper. The double-stained slices were then placed in a copper mesh box to air dry naturally. Images were then observed and analyzed using a transmission electron microscope.

[0091] 2.10. Measurement of cellular ATP content

[0092] Chicken primary hepatocytes were cultured at 5 × 10⁻⁶ 5 Cells were seeded at a density in 12-well plates. When the cells reached 80% confluence, they were treated with FFAs. After 36 hours of FFA treatment, the culture medium in the wells was removed, and the cells were washed twice with 1×PBS. A suitable amount of trypsin was added for digestion, and the cell pellet was collected. The cells were washed again with pre-cooled 1×PBS, centrifuged, and the supernatant was discarded. 1 mL of deionized water was added, and the cells were sonicated (under the following conditions: ice bath, 200W, 5 s interval, 15 s interval, repeated 5 times) to prepare a cell homogenate. ATP content was measured according to the instructions of the kit from Acinetobacter Biotechnology Co., Ltd. The calculation formula is as follows:

[0093]

[0094] 2.11. Measurement of mitochondrial ROS

[0095] 2.11.1. ROS fluorescent staining of mitochondria

[0096] Chicken primary hepatocytes were used at a concentration of 1×10⁻⁶ 6 Cells were seeded at a density in 6-well plates. When the cells reached 80% confluence, they were treated with FFAs. After 36 hours of FFA treatment, the culture medium in the wells was removed, and the cells were washed twice with 1×PBS. 500 μL of mitoSOX Red working solution (working concentration: 2 μM) was added to each well, and the cells were incubated at 37°C for 30 minutes. After incubation, the supernatant was aspirated, and the cells were washed twice with 1×PBS. 500 μL of 1×PBS was added to each well, and the cells were observed and photographed under a fluorescence microscope.

[0097] 2.11.2. Flow cytometry of mitochondria using ROS.

[0098] Chicken primary hepatocytes were used at a concentration of 1×10⁻⁶ 6 Cells were seeded at a density of 100% in 6-well plates. When the cells reached 80% confluence, they were treated with FFAs. After 36 hours of FFA treatment, the culture medium in the wells was removed, and the cells were washed twice with 1×PBS. The cells were then digested with trypsin and collected, washed twice with 1×PBS, centrifuged, and the 1×PBS was discarded. The cells were resuspended in 1 mL of basal culture medium. 500 μL of 2 μM LitoSOX Red working solution was added to each well, and the cells were incubated in a cell culture incubator for 30 min at 37°C. After incubation, the cells were centrifuged (400×g, 4°C, 3 min), and the supernatant was discarded. 1 mL of 1×PBS was added to each well to resuspend the cells, and the cells were washed again (400×g, 4°C, 3 min). The supernatant was discarded, and the washing process was repeated twice. 500 μL of 1×PBS was added to each well to resuspend the cells, and the cells were immediately analyzed by flow cytometry.

[0099] 2.12 Measurement of mitochondrial membrane potential

[0100] 2.12.1. Fluorescent staining of mitochondrial membrane potential

[0101] Chicken primary hepatocytes were used at a concentration of 1×10⁻⁶ 6 Cells were seeded at a density in 6-well plates. When the cells reached 80% confluence, they were treated with FFAs. After 36 hours of FFA treatment, the culture medium in the wells was removed, and the cells were washed twice with 1×PBS. 1 mL of JC-1 working solution (1 mL basal medium + 10 μL JC-1 (100×)) was added to each well. The cells were incubated in a cell culture incubator for 30 minutes at 37°C. After incubation, the supernatant was aspirated, and the cells were washed twice with 1×PBS. 500 μL of 1×PBS was added to each well, and the cells were observed and photographed under a fluorescence microscope.

[0102] 2.12.2 Mitochondrial membrane potential flow cytometry

[0103] Chicken primary hepatocytes were used at a concentration of 1×10⁻⁶6 Cells were seeded at a density of 100% in 6-well plates. When the cells reached 80% confluence, they were treated with FFAs. After 36 hours of FFA treatment, the culture medium in the wells was removed, and the cells were washed twice with 1×PBS. A suitable amount of trypsin was added for digestion, and the cells were collected. The cells were washed twice with 1×PBS, centrifuged, and the 1×PBS was discarded. 1 mL of basal culture medium was added to each well to resuspend the cells. 1 mL of JC-1 working solution (1 mL basal culture medium + 10 μL JC-1 (100×)) was added to each well, and the cells were incubated in a cell culture incubator for 30 min at 37°C. After incubation, the cells were centrifuged (400×g, 4°C, 3 min), and the supernatant was discarded. 1 mL of 1×PBS was added to each well to resuspend the cells, and the cells were washed again by centrifugation (400×g, 4°C, 3 min). The supernatant was discarded, and the washing was repeated twice. 500 μL of 1×PBS was added to each well to resuspend the cells, and the cells were immediately analyzed by flow cytometry. JC-1 monomers were detected in the FITC channel, exhibiting diffuse green fluorescence; JC-1 aggregates were detected in the PI channel, exhibiting dotted red fluorescence.

[0104] 2.13 Protein Extraction and Western Blotting Experiments

[0105] 2.13.1 Total Protein Extraction

[0106] Chicken primary hepatocytes were cultured at 5 × 10⁻⁶ 5 The cells were seeded at a density in 12-well plates. When the cells reached 80% confluence, they were treated with FFAs. After 36 hours of FFA treatment, the culture medium in the wells was removed, and the cells were washed twice with 1×PBS. 500 μL of RIPA lysis buffer containing PMSF was added to each well, and the cells were lysed on ice for at least 30 minutes. The cells were then collected into sterile centrifuge tubes.

[0107] 2.13.2 BCA Concentration Determination

[0108] Protein concentration was determined according to the Solarbio BCA Protein Assay Kit instructions. A standard curve was plotted and the protein concentration of the samples was measured as required. The samples were uniformly diluted, and 20 μL of the sample and 200 μL of BCA working solution were added to each well. The mixture was incubated at 37°C for 30 min, and the OD value was measured at 562 nm using a microplate reader. The concentration of the sample was calculated, and the concentration was standardized using lysis buffer containing PMSF and the phosphatase inhibitor RIPA. The standard curve plotting and sample loading table are shown in Table 7.

[0109] Table 7 Standard Curve Plotting and Sample Addition Table

[0110] Sample hole number 1 2 3 4 5 6 7 (Sample tube) Protein BSA standard (μL) 0 40 80 120 160 200 200 (diluted sample) PBS (μL) 200 160 120 80 40 0 0 BCA working solution (mL) 2 2 2 2 2 2 2

[0111] 2.13.3 SDS-PAGE Adhesive Preparation

[0112] Different concentrations of SDS-PAGE separating gels were prepared according to the different molecular weights of the proteins. The specific preparation methods are shown in Tables 8-9.

[0113] Table 8 SDS-PAGE separating gel preparation

[0114]

[0115] Table 9 SDS-PAGE Stacking Gel Preparation

[0116] Components SDS-PAGE stacking gel (1 sheet of PAGE gel) <![CDATA[ddH2O(mL)]]> 1.4 30% Acr-Bis (mL) 0.33 1.5M Tris pH 8.8 (mL) 0.25 10% SDS (mL) 0.02 10% ammonium persulfate (mL) 0.02 TEMED (mL) 0.002

[0117] 2.13.4. Protein Vertical Electrophoresis and Western Blotting

[0118] (1) Mix the sample with 6× Loading buffer at a ratio of 1:5, boil in boiling water for 10 min, store at -20℃ for later use, vortex mix and centrifuge for 30 s before each use; (2) Remove the comb and add an equal volume of protein sample to each well lane; (3) Electrophoresis at 80V for 40 min; then electrophoresis at 120V for 1 h-1.5 h until bromophenol blue reaches near the bottom of the gel; (4) Transfer membrane: Prepare and pre-cool the transfer buffer in advance, soak the PVDF membrane in methanol to activate it, and place the protein gel, membrane and filter paper in the transfer buffer in a "sandwich" structure. Transfer conditions: constant current 200-400mA; transfer time: depending on the molecular weight of the protein; (5) blocking: place the transferred PVDF membrane in protein blocking solution (rapid blocking solution or 5% skim milk) and block at room temperature for a period of time (block for 15 min with rapid blocking solution; block for 2 h with 5% skim milk); after blocking, repeatedly wash the PVDF membrane with PBST for 30 min (6 min each time, 5 washes); (6) primary antibody incubation: dilute the primary antibody according to the antibody dilution ratio, place the membrane in the primary antibody incubation box and incubate overnight at 4°C. After the primary antibody incubation is completed, repeatedly wash the PVDF membrane with PBST for 30 min (6 min each time, 5 washes); (7) secondary antibody incubation: dilute the secondary antibody according to the antibody dilution ratio, place the membrane in the secondary antibody incubation box and incubate at room temperature for 40 min. After the secondary antibody incubation was completed, the PVDF membrane was repeatedly washed with PBST for 30 min (6 min each time, 5 washes); (8) Exposure and development: According to the instructions of the ultrasensitive ECL chemical development kit (solution A:solution B = 1:1), the PVDF membrane was placed in the development solution for 30 s, and the gel imaging system was used for exposure and photography; (9) Gray value analysis: The obtained exposure bands were statistically analyzed using Image Lab and Image J software, and the corresponding protein gray values ​​were calculated.

[0119] 2.14 Data Statistics and Analysis

[0120] The raw data were initially analyzed using Excel 2019. All data were normalized and presented as mean ± standard deviation (Mean ± SD). In SPSS 26.0, the T-test was used to perform statistical analysis on the differences between the two groups, and one-way ANOVA was used to perform statistical analysis on the differences between more than two groups. A p-value less than 0.05 was considered statistically significant. "*" indicates a comparison with the control group, and "#" indicates a comparison between the two groups. "*" and "#" indicate a p-value less than 0.05, "**" and "##" indicate a p-value less than 0.01, and "***" and "###" indicate a p-value less than 0.001.

[0121] III. Results

[0122] 3.1 Identification, localization, and analysis of lncLRFIF

[0123] This invention, through transcriptomic sequencing of the liver of laying hens with fatty liver hemorrhage syndrome (FLHS), revealed that lncLRFIF (originally named ENSGALT00000095928) is a key molecule regulating lipid metabolism imbalance in the liver. This molecule was significantly highly expressed in the liver tissue of the FLHS model, and its expression level was significantly increased in primary chicken hepatocytes (P<0.05). Therefore, lncLRFIF showed significantly differential expression in the FLHS pathological model constructed using a high-energy, low-protein diet. Figures 15-17 The nucleotide sequence of lncLRFIF is shown in SEQ ID NO.47, as detailed below:

[0124]

[0125] This invention validated the accuracy of lncLRFIF expression patterns in whole transcriptome sequencing data using qPCR experiments. Figure 1 (A and B). RNA located in the cell nucleus and cytoplasm performs different functions and participates in different regulatory mechanisms. Therefore, the nucleoplasmic localization of RNA in the cell is important for predicting and exploring its function. This invention verifies through nucleoplasmic isolation PCR and qPCR that lncLRFIF is expressed in both the cell nucleus and cytoplasm, but is mainly localized in the cytoplasm. Figure 1 (C and D in the middle).

[0126] 3.2 Pearson Correlation Analysis of lncLRFIF

[0127] To determine the importance of lncLRFIF in fatty liver hemorrhage syndrome (FLHS) in laying hens, this invention conducted a correlation analysis between lncLRFIF and basic physiological indicators related to FLHS. Pearson correlation analysis of lncLRFIF confirmed that lncLRFIF was significantly positively correlated with the comprehensive score of fatty liver in laying hens, body weight, blood glucose, NEFA, and ALT (P<0.05), and positively correlated with liver fat percentage, T-CHO, and LDL-C; and significantly negatively correlated with HDL-C (P<0.05), suggesting that lncLRFIF is a key regulator of FLHS. Figure 2 ).

[0128] 3.3 Establishment of a chicken primary hepatocyte lncLRFIF knockdown model

[0129] To further investigate the effects of lncLRFIF on lipid metabolism in chicken primary hepatocytes, this invention synthesized lncLRFIF-specific interfering fragment siRNA (Table 7) and explored suitable transfection concentrations. At a transfection concentration of 50 nmol, there were no significant differences among the four groups: siNC, Gallus-silncLRFIF1 (silncLRFIF1), Gallus-silncLRFIF2 (silncLRFIF2), and Gallus-silncLRFIF3 (silncLRFIF3). At a transfection concentration of 100 nmol, compared with the siNC group, the lncLRFIF expression level in the silncLRFIF3 group was significantly increased (P<0.001). At a transfection concentration of 150 nmol, compared with the siNC group, the lncLRFIF expression level in the silncLRFIF1 group was significantly decreased (P<0.01), while the lncLRFIF expression level in the silncLRFIF3 group was significantly increased (P<0.001). Therefore, this invention selects silncLRFIF1, with a transfection concentration of 150 nmol ( Figure 3 ).

[0130] 3.4 Effect of knockdown of lncLRFIF on damage to chicken primary hepatocytes under FFA treatment

[0131] To investigate the effect of knockdown of lncLRFIF on damage to primary chicken hepatocytes treated with FFAs, this invention transfected silncLRFIF1. Compared with the siNC group, the activities of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) were significantly increased in the FFAs group (P<0.01); compared with the FFAs group, the activities of ALT and AST were significantly decreased in the FFAs+silncLRFIF group (P<0.05). Figure 4 ).

[0132] 3.5 Effect of knockdown of lncLRFIF on lipid metabolism in chicken primary hepatocytes treated with FFAs

[0133] 3.5.1 Effect of knockdown of lncLRFIF on TG (triglycerides) and T-CHO (total cholesterol) levels in chicken primary hepatocytes treated with FFAs

[0134] To investigate the effect of lncLRFIF knockdown on lipid metabolism in chicken primary hepatocytes treated with FFAs, this invention examined the TG and T-CHO contents of chicken primary hepatocytes treated with FFAs after lncLRFIF knockdown. Compared with the siNC group, the TG and T-CHO contents in the FFAs group were significantly increased (P<0.01); compared with the FFAs group, the TG and T-CHO contents in the FFAs+silncLRFIF group were significantly decreased (P<0.05). Figure 5 ).

[0135] 3.5.2 Oil Red Staining and Quantitative Analysis

[0136] Oil Red staining and quantitative analysis of primary chicken hepatocytes showed that, compared with the siNC group, the lipid droplet area was significantly increased in the FFAs group and the FFAs+silncLRFIF group (P<0.001); compared with the FFAs group, the lipid droplet area was significantly decreased in the FFAs+silncLRFIF group (P<0.001). Figure 6 ).

[0137] 3.5.3 Effect of knockdown of lncLRFIF on the expression levels of lipid metabolism-related proteins in chicken primary hepatocytes treated with FFAs

[0138] To investigate the effect of knockdown of lncLRFIF on the expression levels of lipid metabolism-related proteins in chicken primary hepatocytes treated with FFAs, this invention detected the expression levels of these proteins. Compared with the siNC group, the expression level of APOA1 protein in the FFAs group was significantly decreased (P<0.001), while the expression levels of ACC1, SREBP1, and PPARγ proteins were significantly increased (P<0.05). Compared with the FFAs group, the expression level of APOA1 protein in the silncLRFIF group and the FFAs+silncLRFIF group was significantly increased (P<0.01), while the expression levels of ACC1, SREBP1, and PPARγ proteins were significantly decreased (P<0.05). Figure 7 ).

[0139] 3.6 Effect of knockdown of lncLRFIF on mitochondrial energy metabolism in chicken primary hepatocytes treated with FFAs

[0140] 3.6.1 Effect of knockdown of lncLRFIF on ATP (adenosine triphosphate) content in chicken primary hepatocytes treated with FFAs

[0141] To investigate the effect of lncLRFIF knockdown on mitochondrial energy metabolism in chicken primary hepatocytes treated with FFAs, this invention measured the ATP content of chicken primary hepatocytes treated with FFAs after lncLRFIF knockdown. Compared with the siNC group, the ATP content in the FFAs group was significantly decreased (P<0.001); compared with the FFAs group, the ATP content in the silncLRFIF group and the FFAs+silncLRFIF group was significantly increased (P<0.001). Figure 8 ).

[0142] 3.6.2 Effect of knockdown of lncLRFIF on ROS (reactive oxygen species) in chicken primary hepatocytes treated with FFAs

[0143] This invention also examined the mitochondrial ROS levels in hepatocytes after knockdown of lncLRFIF. Compared with the siNC group, the FFA group showed a significant increase in intracellular mitochondrial ROS red fluorescent spots; compared with the FFA group, the silncLRFIF group and the FFA+silncLRFIF group showed a significant decrease in intracellular mitochondrial ROS red fluorescent spots. Figure 9 ).like Figure 10 Flow cytometry analysis showed that, compared with the siNC group, the intracellular mitochondrial ROS level in the FFAs group was significantly increased (P<0.001); compared with the FFAs group, the intracellular mitochondrial ROS level in the silncLRFIF group and the FFAs+silncLRFIF group was significantly decreased (P<0.001).

[0144] 3.6.3 Effect of knockdown of lncLRFIF on mitochondrial membrane potential of chicken primary hepatocytes treated with FFAs

[0145] This invention also detected the membrane potential of mitochondria in chicken primary hepatocytes treated with FFAs after knockdown of lncLRFIF. Compared with the siNC group, the FFA group showed a significant decrease in red fluorescent spots and a significant increase in green fluorescent spots; compared with the FFA group, the FFA+silncLRFIF group showed an increase in red fluorescent spots and a decrease in green fluorescent spots. Figure 11 ).like Figure 12 Flow cytometry analysis showed that, compared with the siNC group, the percentage of cells with low mitochondrial membrane potential was significantly increased in the FFAs group and the FFAs+silncLRFIF group (P<0.001), indicating a significant decrease in mitochondrial membrane potential; compared with the FFAs group, the percentage of cells with low mitochondrial membrane potential was significantly decreased in the FFAs+silncLRFIF group (P<0.01), indicating a significant increase in mitochondrial membrane potential.

[0146] 3.6.4 Effect of knockdown of lncLRFIF on the expression levels of mitochondrial function-related proteins in chicken primary hepatocytes treated with FFAs

[0147] This invention detected the expression levels of mitochondrial function-related proteins in chicken primary hepatocytes treated with FFAs after knockdown of lncLRFIF. Compared with the siNC group, the expression levels of TFAM and IMMT proteins in the FFAs group were significantly decreased (P<0.05); compared with the FFAs group, the expression levels of TFAM and IMMT proteins in the FFAs+silncLRFIF group were significantly increased (P<0.05). Figure 13 ).

[0148] 3.6.5 Effect of knockdown of lncLRFIF on expression levels of mitochondrial respiratory chain-related proteins in chicken primary hepatocytes treated with FFAs

[0149] This invention also detected the expression levels of mitochondrial respiratory chain-related proteins in chicken primary hepatocytes treated with FFAs after knockdown of lncLRFIF. Compared with the siNC group, the expression levels of ATP5B, MTCO2, UQCRC2, SDHB, and NUDUFA9 proteins in the FFAs group were significantly decreased (P<0.05); compared with the FFAs group, the expression levels of ATP5B, MTCO2, UQCRC2, SDHB, and NUDUFA9 proteins in the FFAs+silncLRFIF group were significantly increased (P<0.05). Figure 14 ).

[0150] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An lncLRFIF, characterized in that, The nucleotide sequence of the lncLRFIF is shown in SEQ ID NO.

47.

2. A drug for the prevention and / or treatment of fatty liver hemorrhage syndrome in laying hens, characterized in that, The drug includes an agent that inhibits the expression level of lncLRFIF as described in claim 1; The formulation is Gallus-silncLRFIF1; the nucleotide sequence of the sense strand of Gallus-silncLRFIF1 is shown in SEQ ID NO.41, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.

42.

3. The drug according to claim 2, characterized in that, The drug achieves the effect of preventing and / or treating fatty liver hemorrhage syndrome in laying hens by increasing ALT and AST activity, reducing lipid droplet accumulation, reducing TG and T-CHO content, reducing the expression of lipid synthesis proteins, increasing the expression of lipid transport proteins, increasing ATP and ROS content, and increasing the expression of mitochondrial function-related proteins and mitochondrial respiratory chain-related proteins. The lipid synthesis proteins include ACC1 protein, SREBP1 protein, and PPARγ protein. The lipid transporter includes the APOA1 protein.

4. The drug according to claim 2, characterized in that, The drug also includes pharmaceutically acceptable excipients.

5. The use of lncLRFIF as a target in screening drugs for the prevention and / or treatment of fatty liver hemorrhage syndrome in laying hens, as described in claim 1.

6. A method for screening drugs for treating fatty liver hemorrhage syndrome in laying hens, characterized in that, The method includes the step of detecting the expression level of chicken lncLRFIF before and after drug administration; the nucleotide sequence of the lncLRFIF is shown in SEQ ID NO.47.

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