Preparation of plantain polysaccharide and its application in the preparation of drugs for preventing and treating fatty liver hemorrhagic syndrome in laying hens

Abstract

The present invention discloses the preparation of plantain polysaccharide and its application in the preparation of drugs for preventing and treating fatty liver and hemorrhagic syndrome in laying hens, belonging to the field of biomedicine technology. The present invention also provides the application of plantain polysaccharide in the preparation of feed additives for regulating and / or treating fatty liver in laying hens. 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 liver fat synthesis and oxidative catabolism and ferroptosis-related signaling pathways, thereby effectively preventing and treating fatty liver and hemorrhagic syndrome in laying hens induced by high-energy and low-protein diets. The present invention also studies the preventive and therapeutic effects of plantain polysaccharide on egg production and fatty liver in laying hens, and concludes that plantain polysaccharide can increase egg production in laying hens and improve production performance.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, in particular to the preparation of plantain polysaccharide and 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) is a common nutritional and metabolic disease in laying hens, primarily occurring in well-nourished, heavier flocks with low egg production. Currently, there is no specific treatment for FLHS. In the absence of dedicated therapeutic agents and comprehensive prevention and control mechanisms, prevention and control rely primarily on adjustments to feed formulation and animal husbandry management. The impact of FLHS extends far beyond the individual health of laying hens; it poses a serious challenge to the entire poultry industry. It not only impacts laying performance and reduces the economic benefits of poultry farms, but also increases production costs. More seriously, the prevalence of this disease threatens the sustainable development of the poultry industry.

[0003] Plantain polysaccharides have a wide range of biological activities, including potent antioxidant and anti-inflammatory properties. They can effectively neutralize free radicals, reduce oxidative stress, and protect against inflammation. They can also modulate the immune system, enhance immune function, and show potential to inhibit tumor growth. Studies have not yet found any toxic side effects of plantain on livestock, poultry, or 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 use in the preparation of a medicament for preventing and treating fatty liver and 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 a high-energy, low-protein diet through in vitro and in vivo experimental modeling and detection of relevant indicators. The present invention also reduces hepatic fat deposition by regulating the signaling pathways related to hepatic lipid synthesis, oxidative catabolism, and ferroptosis, thereby effectively preventing and treating fatty liver and hemorrhagic syndrome in laying hens induced by a high-energy, low-protein diet.

[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 fat droplet accumulation in chicken livers and alleviating liver damage in chickens.

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

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

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

[0017] This study establishes a fatty liver model in laying hens through in vitro and in vivo experiments, demonstrating that the application of plantain polysaccharides can significantly increase egg production in laying hens and has a good effect in treating fatty liver. The study investigates the preventive and therapeutic effects of plantain polysaccharides on egg production and fatty liver in laying hens, concluding that plantain polysaccharides can increase egg production and improve production performance. Furthermore, by increasing antioxidant enzyme activity and reducing oxidative stress, they inhibit ferroptosis in the cGAS-STING pathway, effectively improving liver lipid deposition in FLHS laying hens and demonstrating a 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 following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

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

[0020] Figure 2Effects of high-energy, low-protein diet and / or plantain 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 in laying hens of each group;

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

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

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

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

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

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

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

[0031] Figure 13 RT-qPCR results of cGAS-STING pathway-related genes in chicken liver tissue;

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

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

[0034] Figure 16 The effects of each group of plantain polysaccharide on the biochemical parameters of chicken liver cells;

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

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

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

[0038] Figure 20 Effects of Plantago asiatica polysaccharide on MDA, GSH and Fe in chicken liver cells 2+ The impact of levels;

[0039] Figure 21 The effect of each group of plantain polysaccharide on mitochondrial membrane potential of chicken hepatocytes;

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

[0041] Figure 23 The effect of each group of plantain polysaccharide on the iron death-related proteins in chicken hepatocytes;

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

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

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

[0045] Figure 27 The effect of each group of plantain polysaccharide on the protein level of chicken hepatocytes induced by Fer-1 and / or FFAs;

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

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

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

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

[0050] Figure 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] Figure 33 Effects of Plantago asiatica polysaccharide on protein levels in chicken hepatocytes induced by SR717 and / or FFAs;

[0052] Figure 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 described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0054] Example 1 Preparation of Plantago polysaccharide

[0055] (1) Preparation of crude polysaccharide extract: The crude polysaccharide extract was obtained from the raw material by water extraction and alcohol precipitation. The detailed steps are as follows: The dried plantain was crushed with a grinder and passed through a 60-mesh sieve. Anhydrous ethanol (solid-liquid ratio 1:10) was added, and the fat-soluble pigments and some impurities were extracted by stirring at room temperature. The mixture was centrifuged at 6000g for 10 minutes, and the precipitate was collected. Pure water (solid-liquid ratio 1:20) was added to the precipitate, and the mixture was extracted in a 60°C water bath for 4 hours. The mixture was centrifuged at 6000g for 10 minutes, and the supernatant extract was collected. The precipitate residue was extracted again according to the same steps. The two extracts were combined, vacuum rotary evaporation was concentrated to 1 / 10 of the original volume, and four times the volume of anhydrous ethanol was added for alcohol precipitation overnight. The precipitated solid was 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 3000 Da 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: Dissolve 5 g of crude polysaccharide sample in 200 ml of pure water. Centrifuge at 10,000 g for 10 min. Pass the supernatant through an ion exchange column at a flow rate of 4 ml / min. Elute using a gradient of pure water, 0.1 M, 0.2 M, and 0.3 M NaCl solutions, collecting 15 ml of eluate in a tube. Concentrate by rotary evaporation to 1 / 5 of the original volume.

[0058] (4) Gel purification: 1 g of the ion exchange purified polysaccharide sample was added to 20 ml of pure water. Centrifuge at 10,000 g for 10 min. The supernatant was separated and purified by gel chromatography column at a flow rate of 1 ml / min. The eluate was eluted with pure water for 1.5 times the column volume, and 10 ml was collected in one tube. All the eluate was collected. The sample was concentrated by rotary evaporation to 1 / 5 of the original volume. The sample was dialyzed for 48 h using a 3000 Da dialysis bag to remove small molecular components. The polysaccharide was obtained by freeze drying.

[0059] Example 2 In vivo animal experiment

[0060] A fatty liver model was established in FLHS laying hens fed a high-energy, low-protein diet (ME 3079.38 kcal / kg, CP 12.72%) for 16 weeks. Plantago polysaccharide (PLP, see Example 1 for details on its preparation method) was added to a basal diet (ME 2738.10 kcal / kg, CP 17.06%) and a high-energy, low-protein diet at varying doses. 120 100-day-old Hy-Line Brown laying hens, free of specific pathogens and of similar weight and egg production (average weight 1.5 kg ± 0.2 kg), were housed in three-tiered cages. The poultry houses were cleaned and fumigated with formaldehyde for three days beforehand. The laying hens were then acclimated under heat lamps for seven days. The hens were randomly divided into four groups: Group I was the control group, fed a basal diet; Group II was the high-energy, low-protein diet (HELP) group, fed a high-energy, low-protein diet; Group III was the low-dose plantain (LPLP) group (250 mg / kg); Group IV was the high-dose plantain (HPLP) group (500 mg / kg); Group V was the high-energy, low-protein diet combined with 250 mg / kg low-dose plantain (HELP+LPLP); and Group VI was the high-energy, low-protein diet combined with 500 mg / kg high-dose plantain (HELP+HPLP). During the experimental period, the hens were fed feed three times daily (7:00, 12:00, and 17:00) with free access to water. The hens were housed under a 16 / 8 hour light / dark cycle, maintained at a temperature of 18°C-21°C, and a humidity of 40%-70%. The growth and production performance of the hens were observed and recorded. The experimental period lasted 16 weeks, and samples were collected for related experiments.

[0061] After 16 weeks of the experiment, the chickens were fasted for 16 hours, and blood was collected from the wing vein. The blood was then aliquoted into 1.5 ml centrifuge tubes and stored at -20°C. Serum levels of 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) were measured using an automated biochemical analyzer. Liver tissue samples were collected according to grouping. After anesthesia, the chickens were immediately dissected and the livers carefully removed. The weights of the laying hens, liver, and abdominal fat were weighed and recorded. The liver tissues were quickly aliquoted into EP tubes on ice according to grouping and stored at -80°C until further examination. Routine histopathological sections were sectioned, oil red stained, and the liver tissue ultrastructure was observed.

[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 of laying hens in the model of hepatic steatosis 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 the effect was dose-dependent. Figure 2 Compared with the control group, the liver fat rate and abdominal fat content in the HELP group were significantly increased, but decreased significantly after the addition of PLP (P<0.01). At the same time, the liver index increased significantly under HELP induction, but 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, the HELP group had a large number of red lipid droplets in the visual field; the area of ​​red lipid droplets in the visual field in the HELP+LPLP and HELP+HPLP groups was significantly reduced in a dose-dependent manner (P<0.01); lipid droplets in the livers of chickens in the LPLP and HLLP groups were scattered, with centrally located nuclei and clear cell structure, and the area of ​​lipid droplets did not change significantly compared with the control group. These results indicate that PLP can ameliorate HELP-induced lipid droplet accumulation in chicken livers.

[0064] Effects of PLP on the microstructure of liver tissue in laying hens induced by high-energy and low-protein diet Figure 4 As shown in the figure. There were no significant histological changes in the liver tissue structure in the control, LPLP, and HPLP groups. The liver tissue intervals were uniform, and there was no inflammatory cell infiltration. In the HELP group, the liver tissue was swollen, its intact structure was destroyed, and a large number of lipid droplets and vacuoles appeared, accompanied by infiltration of inflammatory factors. Compared with the HELP group, the liver tissue in the HELP+LPLP group was less damaged, its structure was relatively intact, and the number of lipid droplets was significantly reduced. The liver tissue in the HELP+HPLP group showed no significant pathological changes, its tissue structure was intact, and the intervals were uniform. These results indicate that PLP can alleviate HELP-induced liver damage in chickens.

[0065] Serum biochemical indicators: Effects of PLP on serum ALT, AST, TG, T-CHO, LDL, and HDL levels in a high-energy, low-protein diet-induced hepatic steatosis model in laying hens. Figure 5As shown in the figure. Compared with the control group, there were no significant differences in serum ion concentrations in the LPLP and HPLP groups. However, in the HELP group, serum ALT, AST, and LDL levels were significantly increased (P < 0.01), and TG, T-CHO, and HDL levels were significantly increased (P < 0.05). The addition of PLP significantly decreased the levels of all biochemical markers (P < 0.05 and P < 0.01). These results suggest that hepatic steatosis induced by a high-energy, low-protein diet can cause liver damage and disrupt liver lipid metabolism, while PLP effectively mitigates liver damage and regulates liver lipid metabolism.

[0066] Effects of PLP on lipid synthesis-related genes in a high-energy, low-protein diet-induced hepatic steatosis model in laying hens Figure 6 As shown, compared with the control group, the HELP group showed a significant increase in FASN, ACC1, and SREBP-1 mRNA expression (P < 0.01), while PPARα mRNA expression decreased. Compared with the HELP group, both the HELP+LPLP and HELP+HPLP groups significantly decreased FASN, ACC1, and SREBP-1 mRNA expression (P < 0.01), while mitigating the decrease in PPARα mRNA expression. These results suggest that PLP can alleviate lipid deposition by regulating genes related to lipid synthesis.

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

[0068] Effects of PLP on the ultrastructure of liver tissue in a high-energy, low-protein diet-induced hepatic steatosis model 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 laying hen liver induced by high-energy and low-protein diet and restore organelle function. PLP has an effect on MDA, GSH and Fe in the laying hen liver fatty degeneration model induced by high-energy and low-protein diet. 2+ The impact of vitality Figure 9 As shown in the figure, compared with the Control group, the addition of PLP alone reduced the body's oxidative stress and enhanced antioxidant activity. The MDA and Fe 2+ The levels of MDA and Fe in the HELP+LPLP and HELP+HPLP groups were significantly increased (P<0.05), and the levels of GSH were significantly decreased (P<0.01). 2+ The results showed that PLP could effectively reduce liver oxidative damage and regulate iron 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 Steatosis Model Induced by High-Energy and Low-Protein Diet Figure 10 As shown in the results, compared with the control group, the HELP group showed significantly increased mRNA expression levels of NCOA4, ACSL4, and FTH1 (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 decreased the mRNA expression levels of NCOA4, ACSL4, and FTH1 (P<0.05) and upregulated the mRNA expression of GPX4. The HELP+HPLP group significantly alleviated the increased mRNA expression levels of NCOA4, ACSL4, and FTH1, while promoting 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 Figure 11As shown in the results, compared with the control group, the HELP group showed extremely significant increases in NCOA4, ACSL4, and FTH1 protein expression (P<0.01), and an extremely significant decrease in GPX4 protein expression (P<0.01). Compared with the HELP group, the HELP+LPLP group showed extremely significant decreases in NCOA4 and ACSL4 protein expression (P<0.01) and an increase in GPX4 protein expression (P<0.01). The HELP+HPLP group also showed decreased NCOA4, ACSL4, and FTH1 protein expression and an extremely significant alleviation of the decrease in GPX4 protein expression (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 to detect 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 the effect of inhibiting its activation. Figure 12 As shown in Figure 3 , compared with the control group, the HELP group showed a significant increase in cGAS and STING fluorescent foci. Compared with the HELP group, the HELP+LPLP and HELP+HPLP groups showed a dose-dependent decrease in cGAS and STING immunofluorescence foci. These results suggest that PLP may alleviate ferroptosis by inhibiting activation of the cGas-STING pathway.

[0072] Effects of PLP on genes related to the cGAS-STING signaling pathway in a high-energy, low-protein diet-induced hepatic steatosis model in laying hens Figure 13 As shown in the data, 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. 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 Steatosis Model Induced by High-Energy and Low-Protein Diet Figure 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 expression level of TBK1 protein.

[0074] Example 3 In vitro cell test

[0075] The subjects of the in vitro experiment were primary chicken hepatocytes, which were extracted from 12-day-old Hy-Line Gray chicken embryos, and the breeding 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, and sterilize them under UV for 30 minutes. Preheat the type IV collagenase stock 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, remove the chicken embryo and place it in a beaker containing PBS solution. Clean the blood from the chicken embryo, then place the chicken embryo in a beaker, use ophthalmic tweezers to remove the head, feet, and wings, and use another pair of ophthalmic tweezers to open the abdominal cavity from the abdomen. Use a pair of tweezers to remove the liver and place it in a dish containing PBS. Be careful when removing the liver to prevent the gallbladder from being crushed. Cut the liver into pieces of about 1mm 3 Remove small pieces of liver tissue using a Pasteur pipette and transfer them to a centrifuge tube. Wash the liver tissue with PBS until the supernatant is clear. Discard the supernatant and add 5 mL of preheated collagenase solution. Digest at 37°C for 15 minutes. Terminate the digestion with 5 mL of stop culture medium. Filter the digestion solution through a 200-mesh and 500-mesh cell sieve, then centrifuge at low speed to obtain a cell pellet. Resuspend the cells in DMEM medium, centrifuge at low speed to obtain a cell pellet, and resuspend the cells in 5 mL of culture medium. Dissolve 1 μL of the suspension in 3 mL of culture medium, stain with trypan blue, and count viable cells. A final cell density of 1 × 10⁶ cells / mL is used for seeding. Cells are cultured at 37°C with 5% CO₂, with the culture medium replaced every 24 hours. Induce steatosis in chicken hepatocytes using 1 mM FFAs (OA:PA = 2:1).

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

[0077] Chicken hepatocytes were treated with RSL3. The experimental group was divided into five groups: control group, FFA group (1mM FFAs), FFA+RSL3 group (1mM FFAS + 1μM RSL3), FFA+PLP group (1mM FFAs + 5μg PLP), and FFA+RSL3+PLP group (1mM FFAs + 1μM RSL3 + 5μg PLP). Each group was treated with three replicates for 36 hours.

[0078] Chicken hepatocytes were treated with Fer-1. The experimental group was 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 three times, and the cells were treated for 36 hours.

[0079] Chicken hepatocytes were treated with SR717 and divided into five groups: control, FFA (1 mM FFAs), FFA+SR717 (1 mM FFAS + 50 nM SR717), FFA+PLP (1 mM FFAs + 5 μg PLP), and FFA+SR717+PLP (1 mM FFAs + 50 nM SR717 + 5 μg PLP). Each group was treated with three replicates for 36 hours.

[0080] Chicken hepatocytes were treated with C-176. Six experimental groups were assigned: Control, C-176 (1 μM C-176), FFA (1 mM FFAs), FFA+PLP (1 mM FFAs + 5 μg PLP), FFA+C-176 (1 mM FFAs + 1 μM C-176), and FFA+PLP+C-176 (1 mM FFAs + 5 μg PLP + 1 μM C-176). Each group was treated with three replicates for 36 hours.

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

[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 Figure 15 As shown in the results, compared with the control group, hepatocyte lipid droplets in the LPLP and HLPL groups were scattered, with centrally located nuclei and clear cell structures. In the FFA group, numerous red lipid droplets were observed in the visual field, and the lipid droplets became larger, with fusion of lipid droplets visible and an increase in lipid droplet area. Compared with the FFA group, the area of ​​red lipid droplets in the visual field decreased in a dose-dependent manner in the FFA+LPLP and FFA+HPLP groups. These results indicate that PLP can ameliorate FFAs-induced lipid droplet accumulation in chicken hepatocytes.

[0084] Effects of Plantain Polysaccharide on Biochemical Indicators of Chicken Hepatocytes: Effects of PLP on Liver Function Indicators (ALT, AST) and Lipid Metabolism Indicators (TG, T-CHO) in FFA-induced Hepatocyte Fatty Degeneration Model Figure 16 Compared with the control group, the FFA group showed highly significant increases in ALT, AST, TG, and T-CHO levels (P < 0.01). Adding PLP alleviated hepatocellular damage, with significant decreases in TG and T-CHO levels (P < 0.05 and P < 0.01, respectively). These results suggest that PLP effectively mitigates FFA-induced hepatocellular damage and regulates hepatic lipid metabolism.

[0085] Effects of Plantain Polysaccharide on Genes Related to Lipid Synthesis in Chicken Hepatocytes: Effects of PLP on Genes Related to Lipid Synthesis in FFA-induced Hepatocyte Steatosis Model Figure 17As shown in the results, compared with the control group, the FFA group showed a significant increase in the mRNA expression levels of FASN, ACC1, and SREBP-1 (P < 0.05 and P < 0.01), while PPARα mRNA expression decreased. Compared with the FFA group, both the FFA+LPLP and FFA+HPLP groups decreased the mRNA expression levels of FASN, ACC1, and SREBP-1 (P < 0.05 and P < 0.01), while mitigating the decrease in PPARα mRNA expression. These results suggest that PLP can alleviate hepatocyte lipid accumulation by regulating genes related to lipid synthesis.

[0086] Effects of Plantago asiatica polysaccharide on lipid synthesis-related proteins in chicken hepatocytes: Effects of PLP on lipid synthesis-related proteins in the FFAs-induced hepatocyte steatosis model Figure 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 level of PPARα was significantly decreased (P<0.01). Compared with the FFA group, the FFA+LPLP group and the FFA+HPLP group both reduced the mRNA expression levels of FASN, ACC1, and SREBP-1 (P<0.05 and P<0.01), respectively, and alleviated the decrease in PPARα mRNA expression level.

[0087] Effects of Plantain Polysaccharides on Reactive Oxygen Species (ROS) in Hepatocytes: Effects of PLP on ROS Expression in FFAs-induced Hepatocyte Steatosis Model Figure 19 As shown in the results, compared with the control group, the expression of ROS in the FFA group was significantly increased (P<0.01); compared with the FFA group, the expression of ROS in the FFA+LPLP group was reduced, and the expression of ROS in the FFA+HPLP group was significantly reduced (P<0.01). These results indicate that PLP can inhibit the expression 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 FFAs-induced hepatic steatosis model 2+ The impact of vitality Figure 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 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 the levels of MDA and Fe 2+level, increased GSH level, and FFA+HPLP group significantly downregulated MDA and Fe 2+ The results showed that PLP could effectively reduce the oxidative damage of hepatocytes and regulate iron metabolism.

[0089] Effects of Plantain Polysaccharide on Mitochondrial Membrane Potential in Chicken Hepatocytes: Effects of PLP on Mitochondrial Membrane Potential in FFAs-induced Hepatocyte Steatosis Model Figure 21 As shown, the present invention used JC-1 to stain mitochondrial membrane potential. Compared with the control group, JC-1 in the FFA group failed to aggregate effectively, emitting abundant green fluorescence and decreasing mitochondrial membrane potential. 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, emitting red fluorescence and increasing mitochondrial membrane potential. These results indicate that PLP can alleviate mitochondrial damage in hepatocytes.

[0090] Effects of Plantain Polysaccharide on Ferroptosis-Related Genes in Chicken Hepatocytes: Effects of PLP on the Expression of Ferroptosis-Related Genes in FFAs-Induced Hepatocyte Steatosis Model Figure 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 decreased the mRNA expression levels of NCOA4, ACSL4, and FTH1, and significantly increased the mRNA expression of GPX4 (P<0.05). The FFA+HPLP group significantly alleviated the increase in the mRNA expression levels of NCOA4, ACSL4, and FTH1 (P<0.05 and P<0.01), while significantly increasing the mRNA expression of GPX4 (P<0.01).

[0091] Effects of Plantain Polysaccharide on Ferroptosis-Related Proteins in Chicken Hepatocytes: Effects of PLP on the Expression of Ferroptosis-Related Proteins in FFA-induced Hepatocyte Steatosis Model Figure 23 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<0.01), 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 level 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 Figure 24 As shown in the figure, compared with the control group, the FFA group showed a large number of lipid droplets in the visual field, an increase in the number of red lipid droplets in the cytoplasm of chicken hepatocytes, an increase in lipid droplet size, a shift in cell nuclei to one side of the cell, and a significant increase in lipid droplet area (P<0.01). Compared with the FFA group, the FFA+PLP group showed a decrease in the number of lipid droplets in the hepatocytes of chickens, with nuclei returning to the center of the cell, and a significant decrease in the lipid droplet area (P<0.01). Compared with the FFA group, the FFA+RSL3 group showed a large number of lipid droplets in the visual field of chicken hepatocytes, large lipid droplets in the cytoplasm, nuclei squeezed to one side, and blurred nuclei, with a very significant increase in the lipid droplet area ratio (P<0.01). Compared with the FFA+RSL3 group, the FFA+RSL3+PLP group showed a decrease in the number of lipid droplets in the hepatocytes of chickens, a decrease in the number of lipid droplets, a decrease in the size of the lipid droplets, a shift in the nuclei to the center of the cell, and a very significant decrease in the lipid droplet area ratio (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 Figure 25 As shown in the figure, compared with the control group, the FFA group showed extremely significant increases in liver function indicators (ALT and AST), liver lipid metabolism indicators (TG and T-CHO), and lipid peroxidation indicator (MDA), while GSH levels decreased significantly (P<0.05). Compared with the FFA group, the FFA+PLP group showed significant decreases in ALT, AST, and MDA levels (P<0.05 and P<0.01), decreased TG and T-CHO levels, and increased GSH levels. Compared with the FFA+RSL3 group, the FFA+RSL3+PLP group showed significant decreases in ALT, AST, and MDA levels (P<0.05 and P<0.01), decreased TG and T-CHO levels, and increased GSH levels. These results indicate that RSL3 intervention exacerbated FFA-induced hepatocyte damage and lipid deposition in chickens, while PLP improved this phenomenon.

[0094] Effects of Plantain Polysaccharide on the Protein Levels of Chicken Hepatocytes Induced by RSL3 and / or FFAs: Effects of PLP on the Protein Levels of Chicken Primary Hepatocytes Induced by RSL3 and / or FFAs Figure 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 Plantain Polysaccharide on the Protein Levels of Chicken Hepatocytes Induced by Fer-1 and / or FFAs: Effects of PLP on the Protein Levels of Chicken Primary Hepatocytes Induced by Fer-1 and / or FFAs Figure 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 the 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 to detect 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. Figure 28As shown in the figure, compared with the control group, the FFA group showed a significant increase in cGAS and STING fluorescent foci. Compared with the FFA group, the FFA+LPLP and FFA+HPLP groups showed a dose-dependent decrease in cGAS and STING immunofluorescence foci. These results indicate that PLP can inhibit FFAs-induced activation of the cGas-STING pathway in chicken hepatocytes.

[0097] Effects of Plantain Polysaccharide 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 Figure 29 As shown in the results, 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 Polysaccharide on cGAS-STING Pathway-Related Proteins in Chicken Hepatocytes: Effects of PLP on cGAS-STING Signaling Pathway-Related Proteins in FFA-Induced Chicken Primary Hepatocyte Steatosis Model Figure 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 chicken primary hepatocytes induced by SR717 and / or FFAs Figure 31As shown, compared with the control group, the FFA group showed a large number of lipid droplets in the visual field, an increase in the number of red lipid droplets in the cytoplasm of chicken hepatocytes, an increase in lipid droplet size, a shift in cell nuclei to one side of the cell, and a significant increase in lipid droplet area (P < 0.01). Compared with the FFA group, the FFA+PLP group showed a decrease in the number of lipid droplets in the hepatocytes of chickens, with nuclei returning to the center of the cell, and a significant decrease in lipid droplet area (P < 0.05). Compared with the FFA group, the FFA+SR717 group showed a large number of lipid droplets in the visual field of chicken hepatocytes, large lipid droplets in the cytoplasm, nuclei squeezed to one side, and blurred nuclei, and a very significant increase in the lipid droplet area ratio (P < 0.01). Compared with the FFA+SR717 group, the FFA+SR717+PLP group showed a decrease in the number of lipid droplets in the hepatocytes of chickens, a decrease in the size of lipid droplets, a shift in the size of lipid droplets, a shift in the nuclei to the center of the cell, and a very significant decrease in the lipid droplet area ratio (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 Figure 32 As shown in the figure, compared with the control group, the FFA group showed extremely significant increases in liver function indicators (ALT and AST), hepatic lipid metabolism indicators (TG and T-CHO), and lipid peroxidation indicator (MDA) (P<0.05 and P<0.01), and a significant decrease in GSH level (P<0.05). Compared with the FFA group, the FFA+PLP group showed significant decreases in ALT, AST, TG, and MDA levels (P<0.05 and P<0.01), decreased T-CHO levels, and an extremely significant increase in GSH level (P<0.01). Compared with the FFA+RSL3 group, the FFA+RSL3+PLP group showed significant decreases in ALT, AST, and TG levels (P<0.05 and P<0.01), decreased T-CHO and MDA levels, and a significant increase in GSH level (P<0.05). These results indicate that SR717 treatment exacerbated FFA-induced hepatocyte damage and lipid deposition in chickens, while PLP improved this phenomenon.

[0101] Effects of Plantain Polysaccharide on Protein Levels in Chicken Hepatocytes Induced by SR717 and / or FFAs: Effects of PLP on Protein Levels in Chicken Primary Hepatocytes Induced by SR717 and / or FFAs Figure 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 chicken hepatocytes induced by C-176 and / or FFAs: Effects of PLP on protein levels in chicken primary hepatocytes induced by C-176 and / or FFAs Figure 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 merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

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

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