Preparation of plantain herb polysaccharide and application of plantain herb polysaccharide in preparation of medicine for preventing and treating fatty liver hemorrhagic syndrome of laying hens

Abstract

The invention discloses preparation of plantain herb polysaccharide and application of plantain herb polysaccharide in preparation of medicine for preventing and treating fatty liver hemorrhagic syndrome of laying hens, and belongs to the technical field of biological medicine. The invention also provides application of the plantain herb polysaccharide in preparation of a feed additive for regulating and / or treating fatty liver of laying hens. According to the application disclosed by the invention, through in-vivo and in-vitro experimental modeling and detection of related indexes, the prevention and treatment effects of plantain herb polysaccharides with different doses on laying hen fatty liver caused by high-energy low-protein daily ration are comprehensively discussed, and hepatic lipid deposition is reduced by regulating hepatic lipid synthesis and oxidative catabolism as well as ferroptosis related signal channels; therefore, the laying hen fatty liver hemorrhagic syndrome induced by high-energy low-protein daily ration is effectively prevented and treated. The invention also researches the prevention and treatment effects of the plantain herb polysaccharide on the egg yield of laying hens and the fatty liver, and finds that the plantain herb polysaccharide can improve the egg yield of the laying hens and improve the production performance.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to the preparation of plantain polysaccharide and the application of the polysaccharide in the preparation of medicine for preventing and treating fatty liver hemorrhagic syndrome in laying hens. Background Art

[0002] Fatty liver hemorrhagic syndrome (FLHS) in laying hens is a common nutritional metabolic disease in laying hens, which mainly occurs in chickens with good nutrition and large body weight but low egg production rate. There is currently no specific treatment for FLHS. There is a lack of dedicated treatment drugs and a complete prevention and control mechanism. Prevention and control mainly rely on adjusting feed formula and feeding management. The impact of FLHS goes far beyond the individual health of laying hens. FLHS poses a serious challenge to the entire breeding industry. It not only affects the egg-laying performance of laying hens and reduces the economic benefits of farms, but also increases breeding costs. More seriously, the prevalence of this disease threatens the sustainable development of the poultry farming industry.

[0003] Plantain polysaccharides have a wide range of biological activities, including strong antioxidant and anti-inflammatory abilities, which can effectively neutralize free radicals, reduce oxidative stress, and defend against inflammation; at the same time, it can also regulate the immune system, enhance the body's immune function, and show the potential to inhibit tumor growth. Studies have not found any toxic side effects of plantain on livestock and humans, and there are no reports on whether plantain polysaccharides can alleviate FLHS. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation of plantain polysaccharide and its application in the preparation of a drug for preventing and treating fatty liver hemorrhagic syndrome in laying hens, so as to solve the problems existing in the above-mentioned prior art. The present invention comprehensively explores the preventive and therapeutic effects of different doses of plantain polysaccharide on fatty liver in laying hens induced by high-energy and low-protein diets through in vitro and in vivo experimental modeling and detection of relevant indicators, and reduces liver fat deposition by regulating the signal pathways related to liver fat synthesis, oxidative catabolism and ferroptosis, thereby effectively preventing and treating fatty liver hemorrhagic syndrome in laying hens induced by high-energy and low-protein diets.

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

[0006] Technical solution 1: Application of plantain polysaccharide in the preparation of drugs for regulating and / or treating fatty liver in laying hens.

[0007] Technical solution 2: Application of plantain polysaccharide in the preparation of feed additives for regulating and / or treating fatty liver in laying hens.

[0008] Furthermore, the plantain polysaccharide regulates and / or treats fatty liver in laying hens by improving the accumulation of fat droplets in the chicken liver and alleviating the damage to the chicken liver.

[0009] Furthermore, the plantain polysaccharide regulates and / or treats fatty liver in laying hens by alleviating lipid deposition and alleviating mitochondrial damage in chicken liver.

[0010] Furthermore, the plantain polysaccharide regulates and / or treats fatty liver in laying hens by reducing oxidative damage to liver tissue, reducing ferroptosis and inhibiting the expression level of reactive oxygen species.

[0011] Technical solution three: A feed additive, the active ingredient of which includes plantain polysaccharide.

[0012] Technical solution four: A laying hen feed, comprising a basic feed and the feed additive.

[0013] Technical Solution 5: Application of Plantain polysaccharide in the preparation of drugs for preventing and treating fatty liver hemorrhagic syndrome in laying hens induced by high-energy and low-protein diets.

[0014] Technical Solution 6: Application of plantain polysaccharide, the feed additive or the laying hen feed in improving fatty liver of laying hens in the late laying period and improving egg-laying performance.

[0015] Furthermore, the fatty liver of laying hens includes fatty liver of laying hens induced by a high-energy and low-protein diet.

[0016] The present invention discloses the following technical effects:

[0017] The present invention establishes a fatty liver model of laying hens through in vitro and in vivo experiments, and explains that the application of plantain polysaccharide can significantly increase the egg production of laying hens, and also has a good effect on the treatment of fatty liver. The present invention studies the preventive and therapeutic effects of plantain polysaccharide on egg production and fatty liver of laying hens, and obtains the result that plantain polysaccharide can increase the egg production of laying hens and improve production performance. And by increasing the activity of antioxidant enzymes and reducing oxidative stress, the occurrence of ferroptosis in the cGAS-STING pathway is inhibited, thereby effectively improving the level of lipid deposition in the liver of FLHS laying hens, and having a certain protective effect on the liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 Effects of high-energy, low-protein diets and / or psyllium polysaccharides on body weight (A) and egg production (B) of chickens;

[0020] Figure 2Effects of high-energy, low-protein diet and / or psyllium polysaccharide on liver fat percentage (A), abdominal fat percentage (B) and liver index (C);

[0021] Figure 3 Oil red O staining (400×) of chicken livers in each group;

[0022] Figure 4 The changes in the microstructure of the liver tissue of laying hens in each group; H&E staining (200×); the black arrows represent lipid droplets; the red arrows represent inflammatory cell infiltration;

[0023] Figure 5 The changes of serum ALT, AST, TG, T-CHO, HDL and LDL levels of laying hens in each group;

[0024] Figure 6 The RT-qPCR results of lipid synthesis-related genes in chicken liver tissue;

[0025] Figure 7 The results are for the expression of lipid synthesis-related proteins in chicken liver tissue;

[0026] Figure 8 The ultrastructural changes of liver tissues in laying hens in each group; Nucleus: nucleus; Mito: mitochondria; RER: rough endoplasmic reticulum; SER: smooth endoplasmic reticulum; Lip: lipid droplet;

[0027] Fig. 9 Effects of Plantago asiatica polysaccharide on MDA, GSH and Fe in chicken liver tissue 2+ The impact of levels;

[0028] Fig.10 The RT-qPCR results of ferroptosis-related genes in chicken liver tissue;

[0029] Fig.11 The results are for the expression of ferroptosis-related proteins in chicken liver tissue;

[0030] Fig.12 cGAS-STING immunofluorescence of chicken liver tissues in each group, blue fluorescence is cell nuclei, green fluorescence is cGAS protein, and red fluorescence is STING protein (400×);

[0031] Fig.13 The results of RT-qPCR for cGAS-STING pathway-related genes in chicken liver tissue;

[0032] Fig.14 The results are for the expression of cGAS-STING pathway-related proteins in chicken liver tissue;

[0033] Fig.15Oil red O staining of hepatocytes of laying hens in each group (400×);

[0034] Fig.16 The effects of each group of plantain polysaccharide on the biochemical indexes of chicken liver cells;

[0035] Fig.17 RT-qPCR results of lipid synthesis-related genes in chicken hepatocytes;

[0036] Fig.18 The results are for the expression of lipid synthesis-related proteins in chicken hepatocytes;

[0037] Fig.19 The effect of each group of plantain polysaccharide on reactive oxygen species (ROS) in hepatocytes;

[0038] Fig. 20 Effects of Plantago asiatica polysaccharide on MDA, GSH and Fe in chicken hepatocytes 2+ The impact of levels;

[0039] Fig.21 The effects of each group of plantain polysaccharide on mitochondrial membrane potential of chicken hepatocytes;

[0040] Fig. 22 The results of RT-qPCR of each group of plantain polysaccharide on ferroptosis-related genes in chicken hepatocytes;

[0041] Fig.23 The effects of each group of plantain polysaccharide on iron-related death proteins in chicken hepatocytes;

[0042] Fig.24 O staining observation and lipid droplet analysis (400×) of laying hens in each group;

[0043] Fig.25 The effects of each group of plantain polysaccharide on the levels of ALT, AST, TG, T-CHO, MDA and GSH in chicken liver cells;

[0044] Fig.26 Effects of PLP on the expression of FASN, SREBP1, ACSL4, GPX4, cGAS and STING proteins in primary chicken hepatocytes induced by RSL3 and / or FFAs;

[0045] Fig. 27 The effects of each group of plantain polysaccharide on the protein levels of chicken hepatocytes induced by Fer-1 and / or FFAs;

[0046] Fig.28 cGAS-STING immunofluorescence of each group, where blue fluorescence is the cell nucleus, green fluorescence is cGAS protein, and red fluorescence is STING protein (400×);

[0047] Fig.29 RT-qPCR results of cGAS-STING pathway-related genes in chicken hepatocytes;

[0048] Fig.30 The results are for the expression of cGAS-STING pathway-related proteins in chicken hepatocytes;

[0049] Fig.31 Oil red O staining observation and lipid droplet analysis (400×) for each group;

[0050] Fig.32 The effects of each group of plantain polysaccharide on the levels of ALT, AST, TG, T-CHO, MDA and GSH in chicken hepatocytes;

[0051] Fig.33 The effect of Plantago asiatica polysaccharide on the protein level of chicken hepatocytes induced by SR717 and / or FFAs;

[0052] Fig.34 This is the effect of each group of plantain polysaccharides on the protein level of chicken hepatocytes induced by C-176 and / or FFAs. DETAILED DESCRIPTION

[0053] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0054] Example 1 Preparation of Plantago polysaccharide

[0055] (1) Preparation of crude polysaccharide extract: A crude polysaccharide extract is obtained from the raw material by water extraction and alcohol precipitation. The detailed steps are as follows: The dried plantain is crushed with a grinder and passed through a 60-mesh sieve. Anhydrous ethanol (solid-liquid ratio 1:10) is added, and the fat-soluble pigments and some impurities are extracted by stirring at room temperature. The precipitate is centrifuged at 6000g for 10 minutes, and the precipitate is collected. Pure water (solid-liquid ratio 1:20) is added to the precipitate, and the precipitate is extracted in a 60°C water bath for 4 hours. The precipitate is centrifuged at 6000g for 10 minutes, and the supernatant extract is collected. The precipitate residue is extracted again according to the same steps. The two extracts are combined, vacuum rotary evaporation is concentrated to 1 / 10 of the original volume, and four times the volume of anhydrous ethanol is added for alcohol precipitation overnight. The precipitate solid is collected by centrifugation at 8000g for 10 minutes and dried, which is the crude polysaccharide extract.

[0056] (2) Impurity removal (including protein removal, fat removal and decolorization): Add 600 mL of pure water to the crude polysaccharide extract solid to fully dissolve the crude polysaccharide, add 0.4 g of papain, and perform enzymatic hydrolysis overnight. Add 1 / 4 volume of chloroform and n-butanol (4:1, v / v) to the aqueous phase, mix thoroughly, and collect the upper aqueous phase. Add 1 / 4 volume of petroleum ether to the liquid, mix thoroughly, and collect the lower aqueous phase. Add 1 / 2 volume of macroporous resin AB-8 to the aqueous phase, mix thoroughly, and adsorb overnight. Collect the liquid, dialyze it with a 3000Da dialysis bag for 48 hours to remove small molecular components, and precipitate the polysaccharide liquid with alcohol and dry it.

[0057] (3) Ion exchange purification: Take 5g of crude polysaccharide sample and dissolve it in 200ml of pure water. Centrifuge at 10000g for 10min, take the supernatant and purify it through an ion exchange column at a flow rate of 4ml / min; use pure water, 0.1M, 0.2M and 0.3M NaCl solution as gradient elution, collect one tube for every 15ml, and collect all the eluate. Concentrate by rotary evaporation to 1 / 5 of the original volume.

[0058] (4) Gel purification: Take 1g of the polysaccharide sample purified by ion exchange and add 20ml of pure water. Centrifuge at 10000g for 10min, take the supernatant and pass it through a gel chromatography column for separation and purification at a flow rate of 1ml / min; use pure water to elute 1.5 times the column volume, collect one tube for every 10ml, and collect all the eluate. Concentrate by rotary evaporation to 1 / 5 of the original volume. Dialyze for 48h in a 3000Da dialysis bag to remove small molecular components. Freeze-dry to obtain the plantain polysaccharide.

[0059] Example 2 In vivo animal experiment

[0060] The fatty liver model of FLHS laying hens was established with a high-energy, low-protein diet (metabolizable energy 3079.38 kcal / kg, crude protein 12.72%). The experimental period was 16 weeks. Different doses of plantain polysaccharide (preparation method is detailed in Example 1, referred to as PLP) were added to the basal diet (metabolizable energy 2738.10 kcal / kg, crude protein 17.06%) and the high-energy, low-protein diet. 120 100-day-old Hy-Line brown laying hens with no specific pathogens and similar body weight and egg production (average body weight of 1.5 kg ± 0.2 kg) were selected and raised in three-layer cages. The poultry house was cleaned and fumigated with formaldehyde for three days in advance, and the laying hens were adaptively cultured for 7 days under a heat lamp. The chickens were randomly divided into four groups: group I was the control group, which was fed with a basic diet; group II was a high-energy, low-protein diet group (HELP), which was fed with a high-energy, low-protein diet; group III was a 250mg / kg low-dose psyllium group (LPLP); group IV was a 500mg / kg high-dose psyllium group (HPLP); group V was a high-energy, low-protein diet + 250mg / kg low-dose psyllium group (HELP+LPLP); and group VI was a high-energy, low-protein diet + 500mg / kg high-dose psyllium group (HELP+HPLP). During the experiment, the chickens were fed with feed three times a day (7:00, 12:00, and 17:00) and had free access to water. The laying hens were raised in an environment with a light / dark cycle of 16 / 8 hours, with a temperature maintained at 18℃-21℃ and a humidity maintained at 40%-70%. The growth and production performance of the laying hens were observed and recorded. The experimental period was 16 weeks, and samples were collected for related experiments.

[0061] After 16 weeks of the experiment, the chickens were fasted for 16 hours, blood was collected from the wing vein in a centrifuge tube, divided into 1.5 ml centrifuge tubes, and stored at -20 ° C. The serum was tested for alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), total cholesterol (T-Ch), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and total cholesterol (T-CHO) using an automatic biochemical analyzer. Liver tissue samples were collected according to the grouping, and the chickens were dissected immediately after anesthesia, and the liver was carefully removed. The weight of the laying hens, the weight of the liver and abdominal fat were weighed and recorded. The liver tissue was quickly divided into EP tubes on ice according to the grouping and stored at -80 ° C for inspection. Routine pathological histological sections, routine oil red staining of sections and ultrastructural observation of liver tissue were performed.

[0062] The laying hens were weighed and the egg production rate was calculated on the 0th, 30th, 60th, 90th and 120th day of the experiment. The results showed that the effects of plantain polysaccharide (PLP) on the body weight and egg production in the laying hen liver steatosis model induced by high-energy and low-protein diet were as follows: Figure 1As shown in the table, compared with the Control group, the body weight of the HELP group increased significantly, and the egg production decreased significantly (P<0.01); compared with the HELP group, the body weight of the HELP+LPLP group and the HELP+HPLP group decreased significantly, and the egg production increased (P<0.01) on the 60th day, and this was dose-dependent. Figure 2 Compared with the Control group, the liver fat rate and abdominal fat count in the HELP group increased significantly, and decreased significantly after the addition of PLP (P<0.01). At the same time, the liver index increased significantly under HELP induction, and decreased significantly in a dose-dependent manner after PLP treatment (P<0.01).

[0063] Effects of PLP on liver lipid droplets in laying hens induced by high-energy and low-protein diets Figure 3 As shown: compared with the Control group, there were a large number of red lipid droplets in the HELP group; the area of ​​red lipid droplets in the HELP+LPLP and HELP+HPLP groups was significantly reduced in a dose-dependent manner (P<0.01); the lipid droplets in the chicken livers of the LPLP and HLPL groups were scattered, the nucleus was located in the center of the cell, the cell structure was clear, and the lipid droplet area did not change significantly compared with the Control group. The above results indicate that PLP can improve the accumulation of lipid droplets in the chicken liver induced by HELP.

[0064] Changes in the microstructure of the liver tissue of laying hens: Effects of PLP on the liver tissue structure in the model of hepatic steatosis induced by a high-energy, low-protein diet Figure 4 As shown. There were no obvious histological changes in the liver tissue structure of the Control group, LPLP group and HPLP group, the liver tissue intervals were uniform, and there was no inflammatory cell infiltration. The liver tissue of the HELP group was swollen, the complete structure was destroyed, a large number of lipid droplets and vacuoles appeared, and there was infiltration of inflammatory factors; compared with the HELP group, the liver tissue damage in the HELP+LPLP group was milder, the structure was relatively intact, and the number of lipid droplets was significantly reduced; there were no obvious pathological changes in the liver tissue of the HELP+HPLP group, the tissue structure was intact, and the intervals were uniform. The above results show that PLP can alleviate HELP-induced liver damage in chickens.

[0065] Serum biochemical indexes: Effects of PLP on serum ALT, AST, TG, T-CHO, LDL, and HDL levels in the laying hen liver steatosis model induced by high-energy and low-protein diets Figure 5As shown. Compared with the Control group, there was no significant difference in the concentration of each ion in the serum of the LPLP group and the HPLP group, while the levels of ALT, AST, and LDL in the serum of the HELP group increased significantly (P<0.01), and the levels of TG, T-CHO, and HDL increased significantly (P<0.05); after adding PLP, the levels of biochemical indicators decreased significantly (P<0.05 and P<0.01). The results show that hepatic steatosis induced by a high-energy, low-protein diet can cause liver damage and liver lipid metabolism disorders, while PLP can effectively reduce liver damage and regulate liver lipid metabolism.

[0066] Effects of PLP on lipid synthesis-related genes in the laying hen liver steatosis model induced by high-energy and low-protein diet Figure 6 As shown in the figure, compared with the Control group, the mRNA expression levels of FASN, ACC1, and SREBP-1 in the HELP group increased significantly (P<0.01), while the mRNA expression of PPARα decreased. Compared with the HELP group, the HELP+LPLP group and the HELP+HPLP group could significantly reduce the mRNA expression levels of FASN, ACC1, and SREBP-1 (P<0.01), and alleviate the decrease in the mRNA expression level of PPARα. The results show that PLP can alleviate lipid deposition by regulating lipid synthesis-related genes.

[0067] Effects of PLP on lipid synthesis-related proteins in the hepatic steatosis model of laying hens induced by high-energy and low-protein diets Figure 7 As shown in the figure, compared with the Control group, there were no significant differences in the mRNA expression of FASN, ACC1, SREBP-1 and PPARα in the LPLP group and the HPLP group (P>0.05); the mRNA expression levels of FASN, ACC1 and SREBP-1 in the HELP group increased significantly (P<0.01), while the mRNA expression of PPARα decreased significantly (P<0.05). Compared with the HELP group, the HELP+LPLP group and the HELP+HPLP group could significantly reduce the mRNA expression levels of FASN, ACC1 and SREBP-1 (P<0.01), and alleviate the decrease of the mRNA expression level of PPARα (P<0.05).

[0068] Effects of PLP on the ultrastructure of liver tissue in a model of hepatic steatosis induced by a high-energy, low-protein diet in laying hens Figure 8As shown, the mitochondrial structures of the Control group, LPLP group and HPLP group were clear without swelling, the mitochondrial ridges were neatly arranged and complete, and the nuclear structure was clear; compared with the Control group, the mitochondria in the HELP group were severely damaged, the mitochondrial abundance was reduced, the mitochondria were obviously swollen, the mitochondrial ridge structure was blurred and reduced, and lipid droplets appeared in the cytoplasm; compared with the HELP group, the mitochondria in the HELP+LPLP group were less swollen, the number of mitochondria increased, and the mitochondrial membrane boundaries were clear; compared with the HELP group, the mitochondria in the HELP+HPLP group had no obvious pathological changes, the number of mitochondria increased, the mitochondrial ridge structure was clear, and no lipid droplets appeared in the cytoplasm. The results showed that PLP can alleviate the mitochondrial damage in the liver of laying hens caused by high-energy and low-protein diets and restore organelle function. PLP has an effect on the expression of MDA, GSH and Fe in the model of liver fatty degeneration in laying hens induced by high-energy and low-protein diets. 2+ The impact of vitality Fig. 9 As shown in the figure, compared with the Control group, the addition of PLP alone reduced oxidative stress and enhanced antioxidant activity. 2+ Compared with the HELP group, the HELP+LPLP group and the HELP+HPLP group significantly downregulated MDA and Fe 2+ level (P<0.01), and up-regulated GSH level. The results showed that PLP can effectively reduce oxidative damage in liver tissue and regulate iron ion metabolism.

[0069] Effects of Plantain Polysaccharide on Ferroptosis-Related Genes in Hepatic Tissue of Laying Hens: Effects of PLP on Ferroptosis-Related Genes in Hepatic Fatty Degeneration Model of Laying Hens Induced by High-Energy and Low-Protein Diet Fig.10 As shown in the results, compared with the Control group, the mRNA expression levels of NCOA4, ACSL4 and FTH1 in the HELP group were significantly increased (P<0.01), while the mRNA expression level of GPX4 was significantly decreased (P<0.01). Compared with the HELP group, the HELP+LPLP group significantly reduced the mRNA expression levels of NCOA4, ACSL4 and FTH1 (P<0.05), and upregulated the mRNA expression of GPX4. The HELP+HPLP group could significantly alleviate the increase of the mRNA expression of NCOA4, ACSL4 and FTH1, and promote the mRNA expression of GPX4 (P<0.01).

[0070] Effects of Plantain Polysaccharide on Ferroptosis-Related Proteins in Hen Liver Tissue: Effects of PLP on the Expression of Ferroptosis-Related Proteins in Hen Liver Fatty Degeneration Model Induced by High-Energy and Low-Protein Diet Fig.11As shown in the data, compared with the Control group, the protein expression levels of NCOA4, ACSL4, and FTH1 in the HELP group were significantly increased (P<0.01), and the protein expression level of GPX4 was significantly decreased (P<0.01). Compared with the HELP group, the HELP+LPLP group significantly decreased the protein expression levels of NCOA4 and ACSL4 (P<0.01), and upregulated the protein expression level of GPX4 (P<0.01); while the HELP+HPLP group decreased the protein expression levels of NCOA4, ACSL4, and FTH1, and also significantly alleviated the decrease of GPX4 (P<0.01).

[0071] The present invention uses immunofluorescence technology to detect the expression of cGAS and STING, the marker proteins of the cGAS-STING pathway, and detects the activation of the cGAS-STING pathway in the liver tissue of laying hens induced by a high-energy, low-protein diet and whether PLP has an inhibitory effect on its activation. The results are as follows Fig.12 As shown. Compared with the Control group, the cGAS and STING fluorescence clusters in the HELP group increased significantly; compared with the HELP group, the cGAS and STING immunofluorescence clusters in the HELP+LPLP group and the HELP+HPLP group decreased in a dose-dependent manner. The results indicate that PLP may alleviate ferroptosis by inhibiting the activation of the cGas-STING pathway.

[0072] Effects of PLP on cGAS-STING signaling pathway-related genes in the laying hen liver steatosis model induced by high-energy and low-protein diet Fig.13 As shown, compared with the Control group, the HELP group significantly increased the mRNA expression levels of cGAS, STING, IRF3, and TBK1 (P<0.05 and P<0.01). Compared with the HELP group, the HELP+LPLP group downregulated the mRNA expression levels of cGAS, IRF3, and TBK1 (P<0.01), and reduced the mRNA expression level of STING, while the HELP+HPLP group significantly downregulated the mRNA expression levels of cGAS, STING, IRF3, and TBK1 (P<0.05 and P<0.01).

[0073] Effects of Plantain Polysaccharide on cGAS-STING Pathway-Related Proteins in Hen Liver Tissue: Effects of PLP on cGAS-STING Signaling Pathway-Related Proteins in Hen Liver Fatty Degeneration Model Induced by High-Energy and Low-Protein Diet Fig.14As shown in the data, compared with the Control group, the expression levels of cGAS, STING, IRF3, and TBK1 proteins in the HELP group were significantly increased (P<0.01); compared with the HELP group, the HELP+LPLP group extremely significantly downregulated the expression levels of cGAS, STING, IRF3, and TBK1 proteins (P<0.01); the HELP+HPLP group extremely significantly downregulated the expression levels of cGAS, STING, and IRF3 proteins (P<0.01), and downregulated the protein expression level of TBK1.

[0074] Example 3 In vitro cell test

[0075] The subjects of the in vitro experiment were primary hepatocytes of chickens, which were extracted from 12-day-old Hy-Line gray chicken embryos, and the breeder eggs were free of specific pathogens. The incubator was cleaned and disinfected in advance, and the eggs were incubated for 12 days at a humidity of 60% and a temperature of 37.8°C. Preparation: First, place the sterilized surgical instruments, serum-free culture medium, termination culture medium, DMEM culture medium, and PBS buffer on a sterile operating table, sterilize them under ultraviolet for 30 minutes, and preheat the type IV collagenase mother solution in a 37°C water bath. Light an alcohol lamp on the operating table, use tweezers to break the air chamber of the sterilized egg, and then carefully remove the eggshell to expose the air chamber. First, take out the chicken embryo and put it in a beaker containing PBS solution, clean the blood from the chicken embryo, then put the chicken embryo in a beaker, use ophthalmic forceps to remove the head, feet and wings, use another pair of ophthalmic forceps to open the abdominal cavity from the abdomen, and then use a pair of tweezers to take out the liver and put it in a flat dish containing PBS. Be careful when taking the liver to prevent the gallbladder from being broken. Cut the liver into pieces of about 1mm 3 Remove the small pieces of liver tissue from the liver tissue and transfer them into a centrifuge tube with a Pasteur tube. Wash the blood in the liver tissue with PBS until the supernatant is clear. After discarding the supernatant, add 5mL of preheated collagenase liquid and digest at 37℃ for 15min. Use 5mL of termination culture solution to terminate the digestion. The digestion solution is filtered with cell sieves (200 mesh and 500 mesh) in turn, and then centrifuged at a low speed to obtain a cell precipitate. Resuspend the cells with DMEM culture medium, centrifuge at a low speed to obtain a cell precipitate, and add 5mL of culture solution to resuspend the cells. Take 1μL of the suspension and dissolve it in 3mL of culture solution, stain with trypan blue, and count the viable cells. Finally, inoculate at a cell concentration of 1×106 / mL. The cell culture conditions are 37℃ and 5% CO2, and the culture solution is replaced every 24h. Use 1mM FFAs (OA:PA=2:1) ​​to induce fatty degeneration of chicken hepatocytes.

[0076] Chicken hepatocytes were treated with PLP, and the experimental groups were divided into 6 groups: Control group, LPLP group (2.5μg PLP), HPLP group (5μg PLP), FFA group (1mM FFAs), FFA+LPLP group (1mM FFAs+2.5μg PLP), FFA+HPLP group (1mM FFAs+5μg PLP). Each group had 3 replicates, and the cells were treated for 36 hours.

[0077] Chicken hepatocytes were treated with RSL3, and the experimental groups were divided into 5 groups: Control group, FFA group (1mM FFAs), FFA+RSL3 group (1mM FFAS+1μM RSL3), FFA+PLP group (1mM FFAs+5μg PLP), FFA+RSL3+PLP group 1mM FFAs+1μMRSL3+5μg PLP). Each group was repeated 3 times, and the cells were treated for 36 hours.

[0078] Fer-1 was used to treat chicken hepatocytes. The experimental groups were divided into 6 groups: Control group, Fer-1 group (5μM Fer-1), FFA group (1mM FFAs), FFA+PLP group (1mM FFAs+5μg PLP), FFA+Fer-1 group (1mM FFAs+5μM Fer-1), and FFA+PLP+Fer-1 group (1mM FFAs+5μg PLP+5μM Fer-1). Each group was repeated 3 times, and the cells were treated for 36 hours.

[0079] The chicken hepatocytes were treated with SR717 and divided into five groups: Control group, FFA group (1mM FFAs), FFA+SR717 group (1mM FFAS+50nM SR717), FFA+PLP group (1mM FFAs+5μg PLP), and FFA+SR717+PLP group (1mM FFAs+50nM SR717+5μg PLP). Each group was repeated three times, and the cells were treated for 36 hours.

[0080] C-176 was used to treat chicken hepatocytes. The experimental groups were divided into 6 groups: Control group, C-176 group (1μM C-176), FFA group (1mM FFAs), FFA+PLP group (1mM FFAs+5μg PLP), FFA+C-176 group (1mM FFAs+1μM C-176), and FFA+PLP+C-176 group (1mM FFAs+5μg PLP+1μM C-176). Each group was repeated 3 times, and the cells were treated for 36 hours.

[0081] Oil red O staining of hepatocytes was performed; the contents of TG, T-CHO in hepatocytes and ALT and AST in hepatocyte culture medium, MDA and GSH in hepatocytes, and Fe content in hepatocytes were determined according to the instruction manual of Nanjing Jiancheng kit. 2+ content; the intracellular reactive oxygen species (ROS) level was detected by using a reactive oxygen species detection kit; mitochondrial membrane potential MMP was determined; cGAS and STING immunofluorescence staining; the expression level of hepatocyte lipid synthesis gene protein; the expression level of hepatocyte ferroptosis-related gene protein.

[0082] The test data results are analyzed as follows:

[0083] Oil red O staining of chicken hepatocytes: Effects of PLP on lipid droplets in the FFAs-induced hepatocyte steatosis model Fig.15 As shown: Compared with the Control group, the lipid droplets in the hepatocytes of the LPLP and HLPL groups were scattered, the nucleus was located in the center of the cell, and the cell structure was clear. In the FFA group, there were a large number of red lipid droplets in the field of view, the lipid droplets became larger, the lipid droplet fusion phenomenon could be seen, and the lipid droplet area increased; compared with the FFA group, the area of ​​red lipid droplets in the field of view of the FFA+LPLP and FFA+HPLP groups decreased in a dose-dependent manner. The above results indicate that PLP can improve the accumulation of lipid droplets in chicken hepatocytes induced by FFAs.

[0084] Effects of Plantain Polysaccharides on Biochemical Indicators of Chicken Hepatocytes: Effects of PLP on the Levels of Liver Function Indicators (ALT, AST) and Lipid Metabolism Indicators (TG, T-CHO) in the FFAs-induced Hepatocyte Fatty Degeneration Model Fig.16 As shown. Compared with the Control group, the levels of ALT, AST, TG, and T-CHO in the FFA group increased significantly (P<0.01); after adding PLP, liver cell damage was alleviated, and the levels of TG and T-CHO decreased significantly (P<0.05 and P<0.01). The results showed that PLP effectively alleviated FFAs-induced liver cell damage and regulated liver lipid metabolism.

[0085] Effects of PLP on genes related to lipid synthesis in chicken hepatocytes: Effects of PLP on genes related to lipid synthesis in the FFAs-induced hepatocyte steatosis model Fig.17As shown in the figure, compared with the Control group, the mRNA expression levels of FASN, ACC1, and SREBP-1 in the FFA group increased significantly (P<0.05 and P<0.01), while the mRNA expression of PPARα decreased. Compared with the FFA group, the FFA+LPLP group and the FFA+HPLP group could reduce the mRNA expression levels of FASN, ACC1, and SREBP-1 (P<0.05 and P<0.01), and alleviated the decrease in the mRNA expression level of PPARα. The results show that PLP can alleviate hepatocyte lipid deposition by regulating lipid synthesis-related genes.

[0086] Effects of PLP on lipid synthesis-related proteins in chicken hepatocytes: Effects of PLP on lipid synthesis-related proteins in the FFAs-induced hepatocyte steatosis model Fig.18 As shown in the results, compared with the Control group, the mRNA expression levels of FASN, ACC1, and SREBP-1 in the FFA group were significantly increased (P<0.01), while the mRNA expression of PPARα was significantly decreased (P<0.01). Compared with the FFA group, the FFA+LPLP group and the FFA+HPLP group could reduce the mRNA expression levels of FASN, ACC1, and SREBP-1 (P<0.05 and P<0.01), and alleviated the decrease in the mRNA expression level of PPARα.

[0087] Effect of Plantain Polysaccharide on Reactive Oxygen Species (ROS) in Hepatocytes: Effect of PLP on the Expression of ROS in FFAs-induced Hepatocyte Fatty Degeneration Model Fig.19 As shown in the figure, compared with the Control group, the ROS expression in the FFA group was significantly increased (P<0.01); compared with the FFA group, the ROS expression in the FFA+LPLP group was significantly reduced, and the ROS expression in the FFA+HPLP group was significantly reduced (P<0.01). The results showed that PLP can inhibit the expression level of reactive oxygen species.

[0088] Effects of Plantago asiatica polysaccharide on antioxidant enzymes and Fe in chicken hepatocytes 2+ Effect of PLP on MDA, GSH and Fe levels in the FFAs-induced hepatic steatosis model 2+ The impact of vitality Fig. 20 As shown in the figure, compared with the Control group, the addition of PLP alone can enhance the antioxidant activity and reduce Fe 2+ Levels, MDA and Fe in the FFA group 2+ The level of MDA and Fe was significantly increased (P<0.01), and the level of GSH was significantly decreased (P<0.01). Compared with the FFA group, the FFA+LPLP group decreased 2+level, increased GSH level, and FFA+HPLP group significantly downregulated MDA and Fe 2+ The level of PLP was significantly increased (P<0.01), and the level of GSH was significantly increased (P<0.01). The results showed that PLP can effectively reduce the oxidative damage of hepatocytes and regulate iron ion metabolism.

[0089] Effects of PLP on mitochondrial membrane potential in chicken hepatocytes: Effects of PLP on mitochondrial membrane potential in the FFAs-induced hepatocyte steatosis model Fig.21 As shown, the present invention uses JC-1 to stain mitochondrial membrane potential. Compared with the Control group, JC-1 in the FFA group failed to aggregate effectively, emitted abundant green fluorescence, and the mitochondrial membrane potential decreased; compared with the FFA group, JC-1 in the FFA+LPLP and FFA+HPLP groups reaggregated on the mitochondrial membrane in a dose-dependent manner to form J-aggregates, emitted red fluorescence, and the mitochondrial membrane potential increased. The results show that PLP can reduce mitochondrial damage in hepatocytes.

[0090] Effect of Plantain Polysaccharide on Ferroptosis-related Genes in Chicken Hepatocytes: Effect of PLP on the Expression of Ferroptosis-related Genes in FFAs-induced Hepatocyte Steatosis Model Fig. 22 As shown in the results, compared with the Control group, the mRNA expression levels of NCOA4, ACSL4 and FTH1 in the FFA group were significantly increased (P<0.05 and P<0.01), while the mRNA expression level of GPX4 was significantly decreased (P<0.05). Compared with the FFA group, the FFA+LPLP group reduced the mRNA expression levels of NCOA4, ACSL4 and FTH1, and significantly increased the mRNA expression of GPX4 (P<0.05). The FFA+HPLP group could significantly alleviate the increase of the mRNA expression of NCOA4, ACSL4 and FTH1 (P<0.05 and P<0.01), and significantly increase the mRNA expression of GPX4 (P<0.01).

[0091] Effect of Plantain Polysaccharide on Iron Death-Related Proteins in Chicken Hepatocytes: Effect of PLP on the Expression of Iron Death-Related Proteins in FFAs-Induced Hepatocyte Fatty Degeneration Model Fig.23 As shown in the figure, compared with the Control group, the mRNA expression levels of NCOA4, ACSL4 and FTH1 in the FFA group were significantly increased (P<0.01), while the mRNA expression level of GPX4 was significantly decreased (P<0.01). Compared with the FFA group, the mRNA expression levels of NCOA4, ACSL4 and FTH1 in the FFA+LPLP and FFA+HPLP groups were significantly decreased (P<0.01), and the mRNA expression of GPX4 was significantly increased (P<0.01).

[0092] Oil red O staining observation and lipid droplet analysis: Effects of PLP on lipid droplets in chicken primary hepatocytes induced by RSL3 and / or FFAs Fig.24 As shown in the figure, compared with the Control group, there were a large number of lipid droplets in the field of view of the FFA group, the number of red lipid droplets in the cytoplasm of chicken hepatocytes increased, the lipid droplets enlarged, the nucleus was located on one side of the cell, and the area of ​​lipid droplets increased significantly (P<0.01); compared with the FFA group, the number of lipid droplets in the chicken hepatocytes of the FFA+PLP group decreased, the nucleus showed signs of returning to the center of the cell, and the lipid droplet area decreased significantly (P<0.01). Compared with the FFA group, there were a large number of lipid droplets in the field of view of the chicken hepatocytes of the FFA+RSL3 group, large lipid droplets in the cytoplasm, the nucleus was squeezed to one side, the nucleus was blurred, and the lipid droplet area ratio increased significantly (P<0.01); compared with the FFA+RSL3 group, the number of lipid droplets in the chicken hepatocytes of the FFA+RSL3+PLP group decreased, the lipid droplets became smaller, the nucleus returned to the center of the cell, and the lipid droplet area ratio decreased significantly (P<0.01).

[0093] Effects of Plantain Polysaccharide on Biochemical Indices of Chicken Hepatocytes: Effects of PLP on Biochemical Indices of Chicken Primary Hepatocytes Induced by RSL3 and / or FFAs Fig.25 As shown in the figure, compared with the Control group, the liver function indexes (ALT and AST), liver lipid metabolism indexes (TG and T-CHO), and lipid peroxidation index (MDA) in the FFA group were significantly increased (P<0.01), and the GSH level was significantly decreased (P<0.05); compared with the FFA group, the ALT, AST, and MDA levels in the FFA+PLP group were significantly decreased (P<0.05 and P<0.01), the TG and T-CHO levels were decreased, and the GSH level was increased; compared with the FFA+RSL3 group, the ALT, AST, and MDA levels in the FFA+RSL3+PLP group were significantly decreased (P<0.05 and P<0.01), the TG and T-CHO levels were decreased, and the GSH level was increased. The above results show that RSL3 intervention aggravated the FFAs-induced chicken hepatocyte damage and lipid deposition, and PLP improved this phenomenon.

[0094] Effects of PLP on protein levels in primary chicken hepatocytes induced by RSL3 and / or FFAs Fig.26As shown in the figure, compared with the Control group, the levels of lipid synthesis-related proteins (FASN, SREBP1), ferroptosis-related protein (ACSL4), and cGAS-STING pathway-related protein (STING) in the FFA group were extremely significantly increased (P<0.01), the cGAS protein level was increased, and the GPX4 protein level was extremely significantly decreased (P<0.01); compared with the FFA group, the levels of FASN, SREBP1, ACSL4, and STING proteins in the FFA+PLP group were significantly decreased (P<0.01), the cGAS protein level was decreased, and the GPX4 protein level was extremely significantly increased (P<0.01); compared with the FFA+RSL3 group, the levels of SREBP1, ACSL4, and STING proteins in the FFA+RSL3+PLP group were extremely significantly decreased (P<0.01), the levels of FASN and cGASs proteins were decreased, and the GPX4 protein level was significantly increased (P<0.01).

[0095] Effects of PLP on protein levels in chicken hepatocytes induced by Fer-1 and / or FFAs: Effects of PLP on protein levels in primary chicken hepatocytes induced by Fer-1 and / or FFAs Fig. 27 As shown in the figure, compared with the Control group, the levels of lipid synthesis-related proteins (FASN, SREBP1), ferroptosis-related protein (ACSL4), and cGAS-STING pathway-related proteins (cGAS and STING) in the FFA group were extremely significantly increased (P<0.01), and the GPX4 protein level was extremely significantly decreased (P<0.01); compared with the FFA group, the levels of FASN, SREBP1, ACSL4, and STING proteins in the FFA+PLP group and FFA+PLP+Fer-1 group were significantly decreased (P<0.01), the level of cGAS protein was decreased, and the GPX4 protein level was extremely significantly increased (P<0.01); compared with the FFA+PLP group, there were no significant differences in FASN, SREBP1, ACSL4, GPX4, cGAS, and STING in the FFA+Fer-1 group (P>0.05). The above results indicate that PLP can improve FFAs-induced ferroptosis, inhibit the activation of cGAS-STING pathway, and reduce lipid deposition in hepatocytes, just like Fer-1.

[0096] cGAS-STING immunofluorescence: The present invention uses immunofluorescence technology to detect the expression of cGAS and STING, the marker proteins of the cGAS-STING pathway, and detects the activation of the cGAS-STING pathway in primary chicken hepatocytes under the induction of FFAs and whether PLP has the effect of inhibiting its activation. The results are as follows Fig.28As shown. Compared with the Control group, the cGAS and STING fluorescence clusters in the FFA group increased significantly; compared with the FFA group, the cGAS and STING immunofluorescence clusters in the FFA+LPLP group and the FFA+HPLP group decreased in a dose-dependent manner. The results showed that PLP could inhibit the activation of the cGas-STING pathway in chicken hepatocytes induced by FFAs.

[0097] Effects of Plantain Polysaccharides on cGAS-STING Pathway-Related Genes in Chicken Hepatocytes: Effects of PLP on cGAS-STING Signaling Pathway-Related Genes in FFAs-Induced Chicken Primary Hepatocyte Steatosis Model Fig.29 As shown in the data, compared with the Control group, the FFA group significantly increased the mRNA expression levels of cGAS, STING, IRF3, and TBK1 (P<0.05 and P<0.01). Compared with the FFA group, the FFA+LPLP group downregulated the mRNA expression levels of cGAS, STING, IRF3, and TBK1 (P<0.05), while the FFA+HPLP group significantly downregulated the mRNA expression levels of cGAS, STING, IRF3, and TBK1 (P<0.05 and P<0.01).

[0098] Effects of Plantain Polysaccharides on cGAS-STING Pathway-Related Proteins in Chicken Hepatocytes: Effects of PLP on cGAS-STING Signaling Pathway-Related Proteins in FFAs-Induced Chicken Primary Hepatocytes Fatty Degeneration Model Fig.30 As shown in the data, compared with the Control group, the expression levels of cGAS, STING, IRF3, and TBK1 proteins in the FFA group were significantly increased (P<0.05); compared with the FFA group, the FFA+LPLP group downregulated the expression levels of cGAS and STING proteins, and extremely significantly downregulated the expression levels of IRF3 and TBK1 proteins (P<0.01); the FFA+HPLP group extremely significantly downregulated the expression levels of cGAS, STING, and TBK1 proteins (P<0.01), and downregulated the expression level of IRF3 protein.

[0099] Oil red O staining observation and lipid droplet analysis: Effects of PLP on lipid droplets in primary chicken hepatocytes induced by SR717 and / or FFAs Fig.31As shown in the figure, compared with the Control group, there were a large number of lipid droplets in the field of view of the FFA group, the number of red lipid droplets in the cytoplasm of chicken hepatocytes increased, the lipid droplets enlarged, the nucleus was located on one side of the cell, and the lipid droplet area increased significantly (P<0.01); compared with the FFA group, the number of lipid droplets in the chicken hepatocytes of the FFA+PLP group decreased, the nucleus showed signs of returning to the center of the cell, and the lipid droplet area decreased significantly (P<0.05). Compared with the FFA group, there were a large number of lipid droplets in the field of view of the chicken hepatocytes of the FFA+SR717 group, large lipid droplets in the cytoplasm, the nucleus was squeezed to one side, the nucleus was blurred, and the lipid droplet area ratio increased significantly (P<0.01); compared with the FFA+SR717 group, the number of lipid droplets in the chicken hepatocytes of the FFA+SR717+PLP group decreased, the lipid droplets became smaller, the nucleus returned to the center of the cell, and the lipid droplet area ratio decreased significantly (P<0.01).

[0100] Effects of Plantain Polysaccharide on Biochemical Indices of Chicken Hepatocytes: Effects of PLP on Biochemical Indices of Chicken Primary Hepatocytes Induced by SR717 and / or FFAs Fig.32 As shown in the figure, compared with the Control group, the liver function indexes (ALT and AST), liver lipid metabolism indexes (TG and T-CHO), and lipid peroxidation index (MDA) in the FFA group were significantly increased (P<0.05 and P<0.01), and the GSH level was significantly decreased (P<0.05); compared with the FFA group, the ALT, AST, TG, and MDA levels in the FFA+PLP group were significantly decreased (P<0.05 and P<0.01), the T-CHO level was decreased, and the GSH level was significantly increased (P<0.01); compared with the FFA+RSL3 group, the ALT, AST, and TG levels in the FFA+RSL3+PLP group were significantly decreased (P<0.05 and P<0.01), the T-CHO and MDA levels were decreased, and the GSH level was significantly increased (P<0.05). The above results show that SR717 intervention aggravated FFAs-induced chicken hepatocyte damage and lipid deposition, and PLP improved this phenomenon.

[0101] Effects of PLP on protein levels in primary chicken hepatocytes induced by SR717 and / or FFAs Fig.33As shown in the figure, compared with the Control group, the levels of lipid synthesis-related proteins (FASN, SREBP1), ferroptosis-related protein (ACSL4), and cGAS-STING pathway-related proteins (cGAS and STING) in the FFA group were extremely significantly increased (P<0.01), and the GPX4 protein level was significantly decreased (P<0.05); compared with the FFA group, the levels of FASN, SREBP1, ACSL4, and STING proteins in the FFA+PLP group were extremely significantly decreased (P<0.01), the level of cGAS protein was decreased, and the GPX4 protein level was extremely significantly increased (P<0.01); compared with the FFA+SR717 group, the levels of FASN, SREBP1, ACSL4, cGAS, and STING proteins in the FFA+SR717+PLP group were extremely significantly decreased (P<0.01), and the GPX4 protein level was increased.

[0102] Effects of PLP on protein levels in primary chicken hepatocytes induced by C-176 and / or FFAs Fig.34 As shown in the figure, compared with the Control group, the levels of lipid synthesis-related proteins (FASN, SREBP1), ferroptosis-related protein (ACSL4), and cGAS-STING pathway-related proteins (cGAS and STING) in the FFA group were extremely significantly increased (P<0.01), and the GPX4 protein level was extremely significantly decreased (P<0.01); compared with the FFA group, the levels of FASN, SREBP1, ACSL4, cGAS, and STING in the FFA+PLP group and FFA+PLP+Fer-1 group were significantly decreased (P<0.01), and the GPX4 protein level was extremely significantly increased (P<0.01); compared with the FFA+PLP group, there were no significant differences in FASN, SREBP1, ACSL4, GPX4, cGAS, and STING in the FFA+C-176 group (P>0.05). The above results indicate that PLP can inhibit the activation of the cGAS-STING pathway like C-176, improve FFAs-induced ferroptosis, and reduce lipid deposition in hepatocytes.

[0103] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of plantain polysaccharide in the preparation of drugs for regulating and / or treating fatty liver in laying hens.

2. Application of plantain polysaccharide in the preparation of feed additives for regulating and / or treating fatty liver in laying hens.

3. The use according to claim 1 or 2, characterized in that: The plantain polysaccharide regulates and / or treats fatty liver in laying hens by improving the accumulation of fat droplets in the chicken liver and alleviating the damage to the chicken liver.

4. The use according to claim 1 or 2, characterized in that: The plantain polysaccharide regulates and / or treats fatty liver in laying hens by alleviating lipid deposition and relieving mitochondrial damage in chicken liver.

5. The use according to claim 1 or 2, characterized in that: The plantain polysaccharide regulates and / or treats fatty liver in laying hens by reducing oxidative damage to liver tissue and reducing ferroptosis.

6. A feed additive, characterized in that: The active ingredients include psyllium polysaccharide.

7. A laying hen feed, characterized in that: The method comprises a basic feed and the feed additive according to claim 6.

8. Application of plantain polysaccharide in the preparation of drugs for preventing and treating fatty liver hemorrhagic syndrome in laying hens induced by high-energy and low-protein diet.

9. Use of plantain polysaccharide, the feed additive according to claim 6 or the laying hen feed according to claim 7 in improving fatty liver of laying hens in the late laying period and improving egg-laying performance.

10. The use according to claim 9, characterized in that: The fatty liver of laying hens includes fatty liver of laying hens induced by a high-energy and low-protein diet.

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