Application of resveratrol in improving intestinal barrier function and cholesterol metabolism imbalance of cold-exposed broilers

Abstract

The application discloses application of resveratrol in improving intestinal barrier function and imbalance of cholesterol metabolism of cold-exposed broilers and belongs to the technical field of poultry breeding and feed additive. The application finds for the first time that under the condition of cold exposure, resveratrol can significantly reduce the level of cholestenone in the cecal contents of broilers, and up-regulate the expression of genes and proteins related to cholesterol synthesis, transport and intestinal barrier in the liver and intestinal tract, thereby relieving the intestinal barrier damage and cholesterol metabolism disorder caused by cold exposure. The application provides a new use and theoretical basis for developing an anti-cold exposure feed additive.

Description

Technical Field

[0001] This invention belongs to the field of poultry farming technology, specifically relating to the application of resveratrol in improving the intestinal barrier function and cholesterol metabolism imbalance in cold-exposed broilers. Background Technology

[0002] Cold environments are a major stressor restricting the development of poultry farming in high-altitude and cold regions. Cold exposure (ambient temperatures below the animal's optimal survival temperature) induces oxidative stress, inflammatory responses, and metabolic disorders in broilers, significantly impairing growth performance, gut health, and liver function. Among these, disordered cholesterol metabolism in the liver is a significant pathological change caused by cold exposure: decreased expression of the cholesterol synthesis rate-limiting enzyme HMGCR, downregulation of cholesterol efflux-related transport proteins ABCA1 / ABCG1, and abnormal accumulation of cholesterol metabolic intermediates such as cholesterol. Simultaneously, cold exposure disrupts the integrity of the intestinal barrier, leading to decreased expression of tight junction proteins (Claudin, Occludin, ZO-1) and mucin (MUC2), increasing intestinal mucosal permeability. The gut and liver interact closely through the "gut-hepatic axis," and damage to the intestinal barrier exacerbates abnormal liver metabolism.

[0003] Resveratrol is a natural polyphenol compound known to possess antioxidant and anti-inflammatory activities and has been used as a feed additive to improve animal health. However, whether resveratrol can target the interactive damage to the intestinal barrier and cholesterol metabolism under the specific environmental stress of cold exposure, particularly whether it works by regulating cholesterol and gut-hepatic axis-related targets, remains unreported. Summary of the Invention

[0004] In view of this, the present invention aims to provide a novel use of resveratrol in improving intestinal barrier function and cholesterol metabolism imbalance in cold-exposed broilers. This invention is the first to discover that, under specific cold exposure conditions, resveratrol can alleviate intestinal and liver damage caused by cold exposure by reducing cholesterol levels in cecal contents and upregulating the expression of key genes / proteins related to cholesterol synthesis, transport, and the intestinal barrier.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of resveratrol in the preparation of products for improving intestinal barrier function and / or cholesterol metabolism imbalance in cold-exposed broilers.

[0006] Furthermore, the improvement in intestinal barrier function is manifested in: upregulating the mRNA expression of Claudin-1, Claudin-3, TJP-1, and E-cadherin in the cecum, and / or upregulating the protein expression of Occludin, ZO-1, and MUC2.

[0007] Furthermore, the improvement in cholesterol metabolism imbalance is manifested in: upregulating the mRNA expression of SREBF2, HMGCR, ABCA1, ABCG1, INSIG1, and ACAT1 genes in the liver, and / or upregulating the mRNA expression of NPC1L1, PCSK9, ABCG5, ABCG8, and MYLIP genes in the cecum.

[0008] Furthermore, it works by reducing cholesterol levels and via the gut-hepatic axis.

[0009] Furthermore, the cold exposure conditions are an ambient temperature of 8±1℃ for 10 hours per day.

[0010] This invention also provides the application of resveratrol in the preparation of cold-exposure-resistant broiler feed additives or veterinary drug formulations.

[0011] Furthermore, the amount of resveratrol added to feed is 250–750 mg / kg, with the optimal dose being 500 mg / kg.

[0012] It contains at least the following beneficial technical effects: (1) The significant reduction effect of resveratrol on cholesterol in broiler chickens under cold exposure conditions was first clarified, revealing the key metabolite targets for improving cholesterol metabolism.

[0013] (2) The system verified that resveratrol simultaneously repairs the intestinal barrier and restores liver cholesterol metabolism through the gut-liver axis, providing a new mechanism for cold exposure-resistant additives.

[0014] (3) It provides specific cold exposure models, effective dose ranges and detectable effect indicators, which have direct industrial application value. Attached Figure Description

[0015] Figure 1 The effects of cold exposure and Res treatment on broiler growth performance.

[0016] Figure 2 Quality control of intestinal contents metabolite detection.

[0017] Figure 3 PCA analysis charts for each group after cold exposure and Res treatment.

[0018] Figure 4 The PLS-DA analysis score map shows the similarities and differences in the metabolome profiles of each group.

[0019] Figure 5 Clustering heatmap results of differential metabolites in each comparative group after cold exposure and Res treatment.

[0020] Figure 6KEGG pathway analysis in each group after cold exposure and Res treatment.

[0021] Figure 7 Changes in cholesterol-5-en-3-one content in each comparative group after cold exposure and Res treatment.

[0022] Figure 8 Expression levels of cholesterol metabolism-related genes in broiler livers after cold exposure and Res treatment.

[0023] Figure 9 Expression levels of genes and proteins related to cholesterol metabolism in broiler livers after cold exposure and Res treatment.

[0024] Figure 10 Changes in HMGCR protein expression levels in broiler liver tissue.

[0025] Figure 11 Changes in ACAT1 protein expression levels in broiler liver tissue.

[0026] Figure 12 Expression levels of intestinal barrier-related genes in the cecum of broilers after cold exposure and Res treatment.

[0027] Figure 13 Expression levels of cecal barrier-related genes and proteins in broilers after cold exposure and Res treatment.

[0028] Figure 14 Changes in the expression level of MUC2 protein in broiler cecal tissue. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0035] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0036] Example 1 1. Feeding and management of experimental animals A total of 576 one-day-old broiler chickens were selected and randomly assigned to 8 treatment groups: (1) Control group (CON): The feeding temperature was 35℃ from day 1 to day 3, and then decreased by 0.5℃ each day from day 4 to day 32 until it reached 20℃. The animals were fed at this temperature until day 42. They were fed a basic diet throughout the experiment.

[0037] (2) 250 mg / kg resveratrol group (Res250): The feeding temperature was the same as that of the CON group, and the diet supplemented with 250 mg / kg resveratrol was given from day 21 to day 42.

[0038] (3) 500 mg / kg resveratrol group (Res500): The feeding temperature was the same as that of the CON group, and the diet supplemented with 500 mg / kg resveratrol was given from day 21 to day 42.

[0039] (4) 750 mg / kg resveratrol group (Res750): The feeding temperature was the same as that of the CON group, and the diet supplemented with 750 mg / kg resveratrol was given from day 21 to day 42.

[0040] (5) Cold exposure group (CE): The feeding temperature was the same as that of the CON group from day 1 to day 27. Starting from day 28, the group was exposed to cold at 8±1℃ for 10 hours a day (10:00-20:00). The group was fed a basic diet throughout the experiment.

[0041] (6) Cold exposure + 250 mg / kg resveratrol group (CE+Res250): The feeding temperature was the same as that of the CE group, and the feed was supplemented with 250 mg / kg resveratrol from day 21 to day 42.

[0042] (7) Cold exposure + 500 mg / kg resveratrol group (CE+Res500): The feeding temperature was the same as that of the CE group, and the feed was supplemented with 500 mg / kg resveratrol from day 21 to day 42.

[0043] (8) Cold exposure + 750 mg / kg resveratrol group (CE+Res750): The feeding temperature was the same as the CE group, and the diet supplemented with 750 mg / kg resveratrol was fed from day 21 to day 42.

[0044] The main components of the basic feed are as follows: Before 21 days of age: soybean meal 27.00%, DDGS 3.00%, GB26 7.00%, limestone powder 1.00%, dicalcium phosphate 0.90%, salt 0.20%, 70% lysine 0.45%, 98% methionine 0.20%, sodium bicarbonate 0.10%, phytase 5000 0.03%, trace elements 0.12%, broiler compound enzyme preparation 0.02%, broiler compound vitamin 0.03%, 50% choline 0.10%, and mold inhibitor 0.05%; 21-42 days of age: Age: Soybean meal 32.00%, DDGS 4.00%, GB25 8.00%, soybean oil 1.00%, glucose 1.00%, limestone powder 1.20%, dicalcium phosphate 1.40%, salt 0.20%, 70% lysine 0.60%, 98% methionine 0.25%, baking soda 0.10%, phytase 5000 0.04%, trace elements 0.12%, broiler compound enzyme preparation 0.03%, broiler compound vitamin 0.03%, 50% choline 0.10%, antifungal agent 0.05%. Each group of broilers was housed in 6 replicate cages, with 12 chickens per cage. The cage dimensions were 120 cm long × 80 cm wide × 60 cm high (average 800 cm² per chicken). The cages were located in a sterilized experimental chicken house with controlled ambient temperature. During the experiment, the chickens had free access to feed and water and underwent a strict immunization program according to broiler production standards. Check the health status of livestock and poultry daily from 06:00 to 08:00; replenish feed and water; clean feeding equipment; and remove excrement.

[0045] 2. Sample collection and processing At the end of the experiment (day 42), one broiler chicken (n = 6) was randomly selected from each cage and euthanized by cervical dislocation. Serum, liver, cecal tissue, and intestinal contents samples were collected from each group for subsequent analysis. During collection, the liver and cecal tissue samples were divided into two portions: one portion was stored at -80℃ for qRT-PCR and Western blotting, and the other portion was fixed in 4% paraformaldehyde and 2.5% glutaraldehyde electron microscopy fixatives, respectively, for microscopic and submicroscopic morphological observation.

[0046] 3. Growth performance testing The body weight and daily feed intake of each group of broilers were measured and recorded at 29 and 42 days, and the feed conversion ratio of each group of broilers was calculated.

[0047] The effects of cold exposure and Res treatment on broiler growth performance, such as Figure 1 As shown, during the period from the start of cold exposure (29 days) to the end of cold exposure (42 days), there was no significant difference in mean daily weight gain among the CON group, Res250 group, Res500 group, and Res750 group (P>0.05); compared with the CON group, Res250 group, Res500 group, and Res750 group, the mean daily weight gain of the CE group was significantly reduced (P<0.05); the mean daily weight gain of the CE+Res250 group, CE+Res500 group, and CE+Res750 group was higher than that of the CE group, but the difference was not significant (P>0.05). Compared with the CON group, Res250 group, Res500 group, Res750 group, CE+Res250 group, CE+Res500 group and CE+Res750 group, the feed conversion ratio of the CE group was significantly higher (P<0.05); there was no significant difference in the feed conversion ratio of the other groups (P>0.05).

[0048] 4. LC-MS non-targeted metabolomics sequencing analysis 4.1 Extraction of cecal contents Remove the cecal contents sample from the -80℃ freezer and place it on an ice pack to thaw. The specific steps are as follows: (1) Weigh approximately 100 mg of cecal contents from each group of samples (n=6).

[0049] (2) Add 120 µL of 50% methanol solution to each sample, shake the sample to mix it thoroughly, and let it stand at room temperature for 10 min.

[0050] (3) Place the mixture in a -20°C refrigerator overnight to precipitate the protein in the sample.

[0051] (4) Centrifuge at 4000 rpm for 20 min, and transfer the supernatant to a 96-well plate as the metabolite extraction solution.

[0052] (5) Take 10 µL of extract from each sample and mix them in equal amounts to prepare a quality control (QC) sample for subsequent liquid chromatography-mass spectrometry (LC-MS) analysis.

[0053] 4.2 LC-MS Detection and Analysis Metabolites in the samples were separated using an ultra-high performance liquid chromatography (UPLC) system (SCIEX, UK). A TripleTOF 5600 Plus high-resolution tandem mass spectrometer (SCIEX, Warrington, UK) was used to detect the metabolites eluted from the column in both positive and negative ion modes, with a mass scan range of 60 to 1200 Da. The mass spectrometry data were preprocessed using XCMS software, and the results were exported in mzXML format.

[0054] 4.3 Metabolomics Data Analysis Raw LC-MS data underwent peak extraction and annotation using XCMS, CAMERA, and MetaX software. The CAMERA package was used for peak annotation, with each ion identified by its retention time and m / z value. The obtained matrix information was compared with public databases such as KEGG and HMDB, and metabolites were annotated using MetaX software. MetaX software filtered the peak intensity data, selecting those whose characteristic peaks were detected in less than 50% of the QC samples. Simultaneously, the relative standard deviation (RSD) of metabolic characteristics in all QC samples was calculated, and ions with a coefficient of variation greater than 30% (CV>30%) were removed. After quality control, the data were standardized using probability quotient normalization and logarithmic transformation. Subsequently, principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and cluster heatmap analysis were performed on the standardized data to reveal significant differences between different groups. The MetOrigin tool categorizes metabolites into four types: host metabolites, microbial metabolites, co-metabolites (including drug, food, and environment-related metabolites), and other unknown metabolites. Metabolites with significant differences were screened using the Wilcoxon rank-sum test (P<0.05), and metabolic pathway analysis was performed on each metabolite cluster using the enrichment analysis function in MetOrigin.

[0055] Untargeted metabolomics was employed to investigate the effects of cold exposure and Res treatment on cecal metabolism in broilers. Pearson correlation analysis, as shown in Figure 2, reflects the detection status of metabolites in each group. In the figure, the redder the color and the larger the value, the better the sample repeatability and the more stable the instrument's performance throughout the sample analysis. In summary, this metabolomics analysis can detect the types and quantities of metabolites with high quality, enabling accurate quantitative analysis of metabolites.

[0056] PCA analysis results of cold exposure and different concentrations of Res on cecal flora in each group of broilers are as follows: Figure 3 As shown.

[0057] The sample points in the CE group showed a high degree of dispersion, while the CON group, CE group, and CE+Res500 group were clustered into different biological groups.

[0058] The distance between sample points in the CON group and the CE group was relatively large, indicating significant differences. The distance between sample points in the CON group and the CE+Res500 group was also relatively large. Significant differences were observed between the sample points. There was a significant separation between the sample points in the CE group and the CE+Res500 group, which clustered into different biological groups.

[0059] The results of PLS-DA analysis of cecal flora in each group of broilers after cold exposure and treatment with different concentrations of Res are as follows: Figure 4 As shown, PLS-DA analysis can reflect the dispersion and differences between samples. The results show that the distance between samples in the CON group, CE group and CE+Res500 group is large, the similarity between sample points in each group is low, there is no overlapping area, the metabolite composition of the groups changes significantly, and they are clustered into different groups.

[0060] Differences in broiler metabolites among different groups of chickens after cold exposure and treatment with different concentrations of Res are as follows: Figure 5 As shown in the figure. The results indicated that, compared with the CON group, the metabolite composition of the CE group and the CE+Res500 group changed significantly. Cold exposure and Res treatment caused significant changes in the content of different metabolites. Among them, the metabolites 2,4-dimethylthiophene, 3-N-Methylspiperone, uracil, and cholesterol-5-en-3-one were significantly upregulated in the CE group compared with the CON group. P <0.05), Centchroman, Ptaquiloside, and R Metabolites such as Ub schisandrin were significantly downregulated.P <0.05. Compared with the CE group, the CE+Res500 group showed a significant downregulation of metabolites such as octadecanedioate, furamidine, and cholesterol. P <0.05, metabolites such as prostaglandin (PG(PGE1 / i-19:0)), bisnorcholic acid, and mepregnol were significantly upregulated. P <0.05%. Compared with the CON group, the CE+Res500 group showed a significant downregulation of metabolites such as N-acetylsphingosine (C-2 Ceramide), Heterobetulin, and Camelliagenin A. P <0.05%, metabolites such as polyethylene (PE (15:1 / 0:0)), glycerol phosphate (PG (18:1 (11Z) / 18:1 (9Z))) and polyporusterone B were significantly upregulated ( P <0.05). The results of KEGG enrichment analysis of each group of broilers after cold exposure and treatment with different concentrations of Res are as follows: Figure 6 As shown in the figure. The analysis results indicate that, compared with the CE group, the CON group showed enrichment of metabolic pathways, including metabolic pathways, linoleic acid metabolism, arginine biosynthesis, endocytosis, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, and the mTOR signaling pathway. Compared with the CE+Re s500 group, the CE group also showed enrichment of metabolic pathways, including metabolic pathways, linoleic acid metabolism, arginine biosynthesis, endocytosis, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, and the mTOR signaling pathway. Based on HMDB, heatmap, and KEGG results, the metabolites classified as lipids and lipid-like molecules and belonging to metabolic pathways were cholesterols. The cholesterol content was significantly higher in the CE group compared to the CON group. P <0.05%, the cholesterol content in the CE+Res500 group was significantly lower than that in the CE group ( P<0.05), there was no significant difference in cholesterol content between the CON group and the CE+Res500 group ( P >0.05)( Figure 7 ).

[0061] 5. Protein expression level detection 5.1 Protein Extraction (1) Add PMSF to Western blot and IP cell lysis buffer to make its concentration 1 mM.

[0062] (2) Add 1 mL of Western blot blot containing PMSF and IP cell lysis buffer to the grinding tube, take 0.1 g of liver and cecal tissue respectively and place them in the grinding tube and mix well. Place the grinding tube in a cryo-grinding apparatus and grind thoroughly.

[0063] (3) The ground sample was subjected to high-speed frozen centrifugation (4℃, 10000-12000 rpm / min, 5 min), and the supernatant was collected for later use.

[0064] (4) Protein quantification was performed using the BCA method, and the concentrations were adjusted to make the liver protein concentration 9 µg / µL and the cecal protein concentration 5 µg / µL.

[0065] (5) Mix 5 x Buffer and sample at a ratio of 1:4, boil in a boiling water bath for 10 minutes, cool, dispense into portions and store in a -20℃ freezer.

[0066] 5.2 Western Blot (1) Prepare PAGE gel using the PAGE gel rapid preparation kit (Shanghai Yamei Biomedical Technology Co., Ltd., Shanghai, China) according to the instructions. Insert the comb teeth, let stand for 15 min, and then remove the comb teeth.

[0067] (2) Add 4 μL of prestained protein marker to the sample well as an electrophoresis reference. Then, based on the protein concentration calculated by the BCA method, accurately add an appropriate amount of protein sample using a micropipette.

[0068] (3) Adjust the voltage to 150V and electrophoresis for 60 minutes.

[0069] (4) Cut the gel according to the size of the protein, place the gel on a nitrocellulose membrane (NC), and transfer the membrane by adjusting the voltage to 15V for 30 minutes using a semi-dry transfer device (Amersham Biosciences, Boston, US).

[0070] (5) Cut the membrane and wash with TBST buffer for 5 min.

[0071] (6) Pour in skim milk to cover the NC membrane and incubate at 37°C on a shaker for 2 hours. After the incubation period, wash three times with TBST buffer for 5 minutes each time.

[0072] (7) Immerse the NC membrane in the primary antibody dilution solution and incubate overnight at 4°C. See Table 2-3 for protein antibody information.

[0073] (8) Wash with TBST buffer for 5 min, and incubate the NC membrane with IgG secondary antibody (Abclonal, China) in the dark for 60 min.

[0074] (9) Wash with TBST buffer for 5 min and develop the target band using an ECL chemiluminescence kit (Sparklade, China).

[0075] (10) Analyze grayscale values ​​using ImageJ software (NIH, Bethesda, MD). The effects of cold exposure and Res250 on the mRNA expression levels of cholesterol metabolism-related genes in the liver are shown in Figure 8. The mRNA expression levels of SREBF-2, INSIG1, HSP90, HMGCR, ABCA1, and ABCG1 in the CE group were significantly lower than those in the CON, Res250, Res500, and Res750 groups (P<0.05). The mRNA expression level of SREBF-2 in the CE+Res500 and CE+Res750 groups was significantly higher than that in the CE group (P<0.05). The mRNA expression level of INSIG1 in the CE+Res250 and CE+Res500 groups was significantly higher than that in the CE group (P<0.05). The mRNA expression level of HSP90 in the CE+Res250 group was significantly higher than that in the CE group (P<0.05). The mRNA expression levels of HMGCR in the CE+Res250, CE+Res500, and CE+Res750 groups were significantly higher than those in the CE group (P<0.05). The mRNA expression levels of ABCA1 in the CE+Res500 and CE+Res750 groups were significantly higher than those in the CE group (P<0.05). The mRNA expression levels of ABCG1 in the CE+Res750 group were significantly upregulated compared to the CE group (P<0.05). The mRNA expression level of ACAT1 in the CE group was significantly lower than that in the CON group, while the mRNA expression levels of liver ACAT1 in the CE+Res250, CE+Res500, and CE+Res750 groups were significantly higher than those in the CE group (P<0.05).

[0076] The effects of cold exposure and Res on the expression levels of cholesterol metabolism-related genes and proteins in the liver are shown in Figure 9. Compared with the CON group, the expression levels of SREBF and ABCA1 proteins in the livers of broilers were significantly increased in the Res250, Res500, and Res750 groups (P<0.05), with the Res500 group showing the most significant increase. Compared with the control group, the expression levels of HMGCR and ABCA1 proteins were significantly decreased in the CE group (P<0.05). Compared with the CE group, the expression levels of SREBF, HMGCR, and ABCA1 proteins were significantly increased in the CE+Res250, CE+Res500, and CE+Res750 groups (P<0.05).

[0077] Effects of cold exposure and Res on the expression of immunofluorescent proteins in liver cholesterol metabolism, such as Figure 10 As shown in Figure 11, there was no significant difference in HMGCR protein expression levels between the CON group and the Res750 group.P> 0.05), the Res250 and Res500 groups were significantly lower than the CON group ( P <0.05. Compared with the CON group, Res250 group, Res500 group and Res750 group, the expression level of HMGCR protein in the liver of broiler chickens in the CE group was significantly reduced ( P <0.05), the expression levels of HMGCR protein in the livers of broilers in the CE+Res250, CE+Res500, and CE+Res750 groups were significantly increased compared to the CE group. P <0.05), its The increase was most pronounced in the CE+Res750 group. The ACAT1 protein expression level in the liver of broilers in the Res500 group was significantly higher than that in the CON group. P <0.05), the expression level of ACAT1 protein in the CE group was significantly lower than that in the CON group, Res250 group, Res500 group and Res750 group. P <0.05), the expression levels of ACAT1 protein in the CE+Res250 group, CE+Res500 group, and CE+Res750 group were significantly increased compared with the CE group ( P <0.05), with the CE+Res500 group showing the most significant increase.

[0078] The effects of cold exposure and Res on the mRNA expression levels of cecal intestinal barrier-related genes, such as Figure 12 As shown in the figure. Compared with the CE group, the expression levels of Claudin-1 mRNA in the CON group and the Res250, Res500, and Res750 groups were significantly increased ( P <0.05), in the Res250 and Res500 groups, the expression level of Claudin-1 showed an increasing trend compared with the CON group, but the difference did not reach statistical significance ( P >0.05). The mRNA expression levels of Claudin-1 in the CE+Res250 and CE+Res500 groups were significantly increased compared with the CE group ( P <0.05), the Claudin-1 mRNA level in the CE+Res750 group was higher than that in the CE group, but not significantly. P> 0.05). Compared with the CON group, the mRNA expression levels of Claudin-3 in the Res250, Res500, and Res750 groups were significantly increased ( P<0.05), the mRNA expression level of Claudin-3 in the CE group was significantly reduced ( P <0.05), the mRNA expression levels of Claudin-3 in the CE+Res250, CE+Res500, and CE+Res750 groups were significantly higher than those in the CE group ( P <0.05. There was no significant difference in Claudin-4 mRNA expression levels between the CON group and the Res250, Res500, and Res750 groups. P> 0.05), compared with the CON group, the mRNA expression level of Claudin-4 in the CE group was significantly reduced ( P <0.05), the mRNA expression level in the CE+Res500 group was increased compared with that in the CE group, but the increase was not significant. P> 0.05). The mRNA expression levels of TJP-1 in the Res500 and Res750 groups were significantly increased compared to the CON group ( P <0.05), the mRNA expression level in the CE group was significantly lower than that in the CON group ( P <0.05), compared with the CE group, the CE+Res250 group and the CE+Res500 group TJP-1 transcription levels were significantly upregulated in the CE+Res750 group and the CE+Res750 group. P <0.05) and there was no statistically significant difference compared to the CON group and the Res250 group ( P> 0.05). There was no significant difference in TJP-2 mRNA expression levels between the CON group and the Res250, Res500, and Res750 groups. P> 0.05), and significantly higher than the CE group ( P <0.05), the expression levels of TJP-2 mRNA in the CE+Res250, CE+Res500, and CE+Res750 groups showed an increasing trend compared to the CE group, but this did not reach a statistically significant level. P> 0.05). The mRNA expression level of TJP-3 in the CE group was significantly higher than that in the CON group, Res250 group, Res500 group and Res750 group. P <0.05%, the CE+Res750 group was significantly lower than the CE group ( P <0.05. Compared with the CON group, the mRNA expression level of E-cadherin was significantly reduced in the CE group ( P<0.05), the expression levels of E-cadherin mRNA in the CE+Res250 group and the CE+Res750 group were increased compared with those in the CE group, but the increase was not significant. P> 0.05). The effects of cold exposure and Res on the expression levels of cecal intestinal barrier-related genes and proteins, such as Figure 13 As shown. Compared with the CON group, the protein expression levels of Occludin and ZO-1 in the cecum of broiler chickens were significantly downregulated in the CE group ( P <0.05), the protein expression levels of Occludin in the CE+Res250 group and the CE+Res500 group were significantly higher than those in the CE group ( P <0.05), the protein expression level of ZO-1 in the CE+Res250 group was significantly higher than that in the CE group ( P <0.05).

[0079] Effects of cold exposure and Res on immunofluorescence expression of cecal intestinal barrier proteins, such as Figure 14 As shown. Compared with the CON group, the expression levels of MUC2 protein in the cecum of broiler chickens were significantly increased in the Res250, Res500, and Res750 groups. P <0.05%, with the Res750 group showing the most significant upward trend. The expression level of MUC2 protein in the cecum of broiler chickens in the CE group was significantly lower than that in the CON, Res250, Res500, and Res750 groups. P <0.05), CE+Res250 group, CE+Res500 group and CE+Res750 Compared to the CE group, the expression level of MUC2 protein in the cecum of broiler chickens in the CE group was significantly increased. P <0.05), with the CE+Res750 group showing the most significant upward trend.

[0080] in conclusion: (1) Cold exposure at 8±1℃ will reduce the body weight gain of broilers, cause oxidative stress in the body, lead to abnormal intestinal structure and reduced barrier function, and liver metabolism disorder.

[0081] (2) Non-targeted metabolomics revealed that cold exposure alters the levels of intestinal metabolites in broilers and affects liver metabolism by promoting cholesterol production and inhibiting cholesterol metabolism.

[0082] (3) Resveratrol can improve intestinal barrier function by reshaping the balance of intestinal metabolites in cold-exposed broilers, and improve liver cholesterol metabolism abnormalities caused by cold exposure by stimulating the expression of cholesterol synthesis and transport-related regulatory factors.

[0083] Therefore, resveratrol alleviates cold exposure-induced damage to the intestinal and liver tissues of broilers by regulating the balance of intestinal metabolites and based on the gut-liver axis.

[0084] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of resveratrol in the preparation of products for improving intestinal barrier function and / or cholesterol metabolism imbalance in cold-exposed broilers.

2. The application according to claim 1, characterized in that, The improvement in intestinal barrier function is manifested in: upregulating the mRNA expression of Claudin-1, Claudin-3, TJP-1, and E-cadherin in the cecum, and / or upregulating the protein expression of Occludin, ZO-1, and MUC2.

3. The application according to claim 1, characterized in that, The improvement in cholesterol metabolism imbalance is manifested by upregulating the mRNA expression of SREBF2, HMGCR, ABCA1, ABCG1, INSIG1, and ACAT1 genes in the liver, and / or upregulating the mRNA expression of NPC1L1, PCSK9, ABCG5, ABCG8, and MYLIP genes in the cecum.

4. The application according to claim 1, characterized in that, It works by lowering cholesterol levels and via the gut-hepatic axis.

5. The application according to claim 1, characterized in that, The cold exposure conditions are an ambient temperature of 8±1℃ for 10 hours per day.

6. Application of resveratrol in the preparation of cold-resistant broiler feed additives or veterinary drug formulations.

7. The application according to claim 6, characterized in that, The recommended addition level of resveratrol to feed is 250–750 mg / kg.

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