A traditional chinese medicine monomer for treating avian necrotic enteritis and application thereof

Abstract

This invention discloses a traditional Chinese medicine monomer for treating necrotizing enteritis in poultry and its application, belonging to the field of traditional Chinese veterinary medicine technology. This monomer is composed of resveratrol, with a dosage of 125-500 mg per kilogram of feed, preferably 250 mg. The resveratrol of this invention treats necrotizing enteritis in chickens through a synergistic effect of direct in vitro antibacterial activity and in vivo intestinal barrier protection: the MIC against Clostridium perfringens in vitro is 0.16 mg / mL, exerting its antibacterial effect through multiple targets including inhibition of cell wall synthesis, energy metabolism, and virulence regulation; in vivo, it significantly improves the growth performance of NE broilers, reduces intestinal lesion scores, repairs the intestinal barrier, regulates immune inflammatory responses, and enhances antioxidant capacity. This invention provides theoretical and experimental basis for the development of natural control agents against intestinal pathogens in livestock and poultry, possessing the characteristics of high efficiency, safety, and no residue, and providing a new approach to replace antibiotics in the treatment of necrotizing enteritis in chickens.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese veterinary medicine technology, and in particular to a single traditional Chinese medicine monomer for treating necrotizing enteritis in poultry and its application. Background Technology

[0002] Necrotic enteritis, an acute intestinal infectious disease of poultry caused by Clostridium perfringens, is a key bottleneck restricting the healthy development of the global poultry farming industry. Clostridium perfringens, a Gram-positive anaerobic spore-forming bacterium, secretes various virulence factors such as alpha toxin and NetB toxin, which damage the integrity of intestinal mucosal epithelial cells, causing mucosal congestion, hemorrhage, and necrotic lesions. The morbidity rate in broilers can reach 40%, with mortality rates as high as 30%–50% during acute outbreaks. Subclinical infections lead to a significant reduction in feed conversion ratios, resulting in global economic losses exceeding US$2 billion annually.

[0003] For a long time, antibiotics such as bacitracin zinc and virginiamycin have been widely used for the prevention and treatment of necrotizing enterocolitis. However, long-term low-dose addition has led to the emergence of drug-resistant strains of Clostridium perfringens, causing a series of problems such as veterinary drug residues, intestinal microecological imbalance, and environmental pollution. Developing safe, effective, and residue-free antibiotic alternatives has become a research hotspot in the field of poultry nutrition and health.

[0004] Natural plant extracts are rich in various bioactive compounds, such as polyphenols, flavonoids, tannins, terpenes, phenolic acids, saponins, alkaloids, and polysaccharides, which have anti-inflammatory, antioxidant, anti-aging, and anti-tumor effects. Our research team previously screened a compound preparation with significant efficacy against norepinephrine (NE) from traditional Chinese medicine for clearing heat and detoxifying. Through systematic activity tracking and network pharmacology analysis, we identified resveratrol as the key active monomer for the compound's anti-Clostridium perfringens activity.

[0005] Resveratrol is a natural polyphenol compound mainly found in plants such as Polygonum cuspidatum and grapes. Studies have shown that resveratrol possesses various biological activities, including antioxidant, anti-inflammatory, cardiovascular protective, and anti-tumor effects. The chemical structural formula of resveratrol is shown below.

[0006]

[0007] Currently, there are no reports on the application of resveratrol in the treatment of necrotic enteritis in chickens. Therefore, providing information on the application of resveratrol in the preparation of drugs for treating necrotic enteritis in broilers is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a traditional Chinese medicine monomer with low toxicity and side effects, low drug residue, and good efficacy for the treatment of necrotizing enteritis in chickens and its application.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A single monomer of traditional Chinese medicine, composed of resveratrol.

[0011] The present invention also provides an application of a traditional Chinese medicine monomer, wherein the dosage of resveratrol is 125-500 mg of resveratrol per kilogram of feed.

[0012] Furthermore, the resveratrol is used at a dosage of 250 mg per kilogram of feed.

[0013] This invention also provides the application of a traditional Chinese medicine monomer in the preparation of a drug for treating necrotizing enteritis in chickens.

[0014] Furthermore, the application includes its use in the preparation of drugs that enhance the immune function of broilers infected with necrotizing enteritis.

[0015] Furthermore, the application includes its use in the preparation of drugs to improve the growth performance of broilers infected with necrotizing enteritis.

[0016] Furthermore, the application includes its use in the preparation of drugs for repairing the intestinal barrier in broilers infected with necrotizing enterocolitis.

[0017] Furthermore, the application includes its use in the preparation of drugs that enhance the antioxidant properties of broilers infected with necrotizing enteritis.

[0018] Furthermore, the application includes its use in the preparation of drugs that directly inhibit the proliferation of Clostridium perfringens.

[0019] Furthermore, the necrotizing enteritis in chickens was caused by a mixed infection of Eimeria giantiformis and Clostridium perfringens type A.

[0020] Compared with currently available technologies, the innovative achievements of this invention demonstrate the following technical advantages:

[0021] (1) This invention is the first to demonstrate that resveratrol has direct antibacterial activity against Clostridium perfringens, with a minimum inhibitory concentration of 0.16 mg / mL, comparable to that of bacitracin, a commonly used clinical antibiotic. Transcriptomic analysis reveals that resveratrol inhibits Clostridium perfringens through a four-pronged mechanism of "structural synthesis-energy metabolism-protein synthesis-motor regulation": inhibiting cell wall synthesis, blocking energy metabolism, downregulating ribosome pathways and virulence regulatory genes. Simultaneously, resveratrol significantly inhibits the sliding movement and biofilm formation of Clostridium perfringens, thus blocking bacterial invasion and persistent infection at the source.

[0022] (2) This invention demonstrates good therapeutic potential for necrotizing enteritis in chickens. In vivo experiments show that resveratrol not only significantly improves the growth rate of diseased broilers, enhances immune defense mechanisms and antioxidant capacity, but also effectively promotes the recovery of the intestinal barrier after damage. Medium-dose resveratrol significantly improves growth inhibition caused by necrotizing enteritis, reduces intestinal lesion scores and permeability, upregulates tight junction protein expression, downregulates pro-inflammatory factors, and enhances serum antioxidant capacity.

[0023] (3) The innovative traditional Chinese medicine monomer components introduced in this invention, with their high efficiency, safety, no residue and easy application, have opened up a completely new non-antibiotic approach for the treatment of necrotizing enteritis in chickens. This breakthrough has brought significant changes to the field of necrotizing enteritis prevention and control, and provides an innovative technical route to solve the current problem of excessive reliance on antibiotics in this field. Attached Figure Description

[0024] Figure 1 The growth performance graphs for each group are shown below. In the graphs: (A) is the average daily weight gain of each group, (B) is the average daily feed intake of each group, and (C) is the feed conversion ratio of each group. Among them, Con is the blank control group, Mod is the model group, BMD is the serotonin group, LRes is the low-dose resveratrol group, MRes is the medium-dose resveratrol group, and HRes is the high-dose resveratrol group.

[0025] Figure 2 The following are the intestinal necropsy results and intestinal lesion scoring charts for each group. In the chart: (A) is the duodenal lesion score, (B) is the jejunal lesion score, (C) is the ileal lesion score, (D) is the necropsy result of the Con group, (E) is the necropsy result of the Mod group, (F) is the necropsy result of the BMD group, (G) is the necropsy result of the LRes group, (H) is the necropsy result of the MRes group, and (I) is the necropsy result of the HRes group.

[0026] Figure 3 The images show tissue sections of the jejunum and ileum for each group. In the images: (A) is the jejunum of the Con group, (B) is the jejunum of the Mod group, (C) is the jejunum of the BMD group, (D) is the jejunum of the LRes group, (E) is the jejunum of the MRes group, (F) is the jejunum of the HRes group, (G) is the ileum of the Con group, (H) is the ileum of the Mod group, (I) is the ileum of the BMD group, (J) is the ileum of the LRes group, (K) is the ileum of the MRes group, and (L) is the ileum of the HRes group.

[0027] Figure 4 The graph shows the concentration of FITC-d in serum. In the graph, Con is the blank control group, Mod is the model group, BMD is the serotonin group, LRes is the low-dose resveratrol group, MRes is the medium-dose resveratrol group, and HRes is the high-dose resveratrol group.

[0028] Figure 5 The diagram shows the relative expression levels of genes related to the jejunal barrier. In the diagram: (A) is the relative expression level of the ZO-1 gene, (B) is the relative expression level of the Occludin gene, and (C) is the relative expression level of the MUC-2 gene. Among them, Con is the blank control group, Mod is the model group, BMD is the serotonin group, LRes is the low-dose resveratrol group, MRes is the medium-dose resveratrol group, and HRes is the high-dose resveratrol group.

[0029] Figure 6 The figure shows serum antioxidant indicators. In the figure: (A) is peroxidase (CAT), (B) is superoxide dismutase (SOD), and (C) is malondialdehyde (MDA). Among them, Con is the blank control group, Mod is the model group, BMD is the serotonin group, LRes is the low-dose resveratrol group, MRes is the medium-dose resveratrol group, and HRes is the high-dose resveratrol group.

[0030] Figure 7 The diagram shows the relative expression levels of cytokine-related genes in jejunal tissue. In the diagram: (A) is the relative expression level of IL-1β gene, (B) is the relative expression level of IL-6 gene, (C) is the relative expression level of IL-10 gene, and (D) is the relative expression level of TNF-α gene. Among them, Con is the blank control group, Mod is the model group, BMD is the serotonin group, LRes is the low-dose resveratrol group, MRes is the medium-dose resveratrol group, and HRes is the high-dose resveratrol group.

[0031] Figure 8 The graph shows the levels of immunoglobulins and necrotizing enterocolitis markers in jejunal tissue. In the graph: (A) is calprotectin, (B) is IgA, (C) is IgM, and (D) is IgY. Among them, Con is the blank control group, Mod is the model group, BMD is the serotonin group, LRes is the low-dose resveratrol group, MRes is the medium-dose resveratrol group, and HRes is the high-dose resveratrol group. Detailed Implementation

[0032] 1. Method

[0033] 1.1 Experimental Materials

[0034] 144 one-day-old male broiler chickens were purchased from Guangxi Dongshi Agricultural and Animal Husbandry Co., Ltd. Eimeria giant coccidia oocysts were provided by the Traditional Chinese Veterinary Medicine Laboratory of the College of Animal Science and Technology, Guangxi University. Clostridium perfringens type A CVCC2030 was purchased from the China Veterinary Microbial Culture Collection Center. Resveratrol (≥98%, CAS: 501-36-0) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Methylene salicylate bacitracin soluble powder (Sulfadiazine) was purchased from Greencon Biochemical Co., Ltd. Dextran-fluorescein isothiocyanate (FITC-d, MW 4000) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. TRIzol® Reagent, StarScript III one-tube genomic de-genome reverse transcription premix, and 2×RealStar Fast SYBR qPCR Mix were all purchased from Kangrun Jingxing (Suzhou) Biotechnology Co., Ltd.

[0035] 1.2 Preparation of giant Eimeria coccidia oocysts

[0036] Eimeria giantiformis oocysts were obtained from the Traditional Chinese Veterinary Laboratory of the College of Animal Science and Technology, Guangxi University. Sporulated oocysts of Eimeria giantiformis stored at 4℃ were passaged in 14-day-old coccidia-free broiler chickens. Purification, sporulation, and oocyst counting of Eimeria giantiformis were performed according to standard methods. To induce necrotizing enteritis caused by Clostridium perfringens, purified Eimeria giantiformis were stored at 4℃ for no more than one month to ensure oocyst viability.

[0037] 1.3 Preparation of Clostridium perfringens strains

[0038] Clostridium perfringens type A CVCC2030 was purchased from the China Veterinary Microbial Culture Collection Center. CVCC2030 was removed from cryopreservation tubes and inoculated onto meat broth. After overnight incubation at 37°C, the culture was streaked onto TSC medium and anaerobically cultured for 24 hours. Black single colonies were picked and re-inoculated onto meat broth, cultured to the logarithmic growth phase, and stored for later use. The reserved bacterial culture was diluted 1:1000 into fresh meat broth and anaerobically cultured at 37°C for 11 hours. The resulting fresh culture was centrifuged at 4000 rpm for 10 minutes at 4°C to collect the cells. The cells were washed three times with sterile PBS, and the bacterial concentration was adjusted with PBS. The bacterial concentration was determined to be 1 × 10⁻⁶ using the plate count method. 9 CFU / mL.

[0039] 1.4 In vitro antibacterial test

[0040] 1.4.1 Determination of Minimum Inhibitory Concentration (MIC)

[0041] After thawing the frozen Clostridium perfringens CVCC2030, it was activated on meat broth and passaged to the logarithmic growth phase (10⁻¹⁰).9 CFU / mL). Inoculate into MH broth at a ratio of 1:1000 and anaerobic culture at 37°C until the bacterial concentration reaches 1×10⁻⁶. 5 CFU / mL. MIC was determined using the microbroth dilution method: 1 mL of MH broth was added to a sterile test tube, followed by 1 mL of resveratrol stock solution (5 mg / mL) to the first tube. This was serially diluted 2-fold up to the 11th tube, with the 12th tube serving as a blank control. 100 μL of bacterial culture (final concentration 1 × 10⁻⁶) was added to each tube. 5 (CFU / mL), anaerobic culture at 37℃ for 16 h. MIC is defined as the lowest drug concentration at which no bacterial growth is observed. A DMSO solvent control (final concentration ≤1%, confirming no antibacterial activity) and a bacitracin control (positive control) were also included.

[0042] 1.4.2 Sliding motion inhibition test

[0043] Collect *Clostridium perfringens* in the logarithmic growth phase, centrifuge at 4000 rpm for 10 min to collect the cells, wash three times with sterile PBS, resuspend in BHI semi-solid medium (containing 0.3% agar), and adjust the bacterial concentration to 1×10⁻⁶. 6 CFU / mL. 10 μL of bacterial suspension was vertically added to the center of BHI plates containing different concentrations of resveratrol (0, 0.08, 0.16, 0.32 mg / mL), and the plates were anaerobically cultured at 37°C for 72 h. The diameter of the sliding zone was measured, and the inhibition rate was calculated.

[0044] 1.4.3 Biofilm Formation Inhibition Test

[0045] Adjusting Clostridium perfringens to OD 600 =0.1 (approximately 1×10) 6 CFU / mL), 100 μL was added to a 96-well plate, along with different concentrations of resveratrol (0.078125-2.5 mg / mL, serially diluted 2 times), with 6 replicates per group. After anaerobic incubation at 37℃ for 48 h, the supernatant was discarded, and the plate was gently washed 3 times with PBS and dried at 37℃ for 1 h. 200 μL of 0.1% crystal violet was added for staining for 15 min, followed by washing with PBS to remove excess stain. 200 μL of 33% acetic acid was added to dissolve the crystal violet, and the OD was measured. 570 Zero the biofilm inhibition rate using the blank well.

[0046] 1.5 Prokaryotic Transcriptome Sequencing Analysis

[0047] 1.5.1 Bacterial Processing and Sample Preparation

[0048] Clostridium perfringens was diluted 1:100 in fresh RCM medium and anaerobically cultured at 37°C for 6 h until the logarithmic growth phase. Resveratrol was added to a final concentration of 0.16 mg / mL (MIC value), and the culture was continued for 3 h. The bacterial cells were collected, centrifuged at 4°C and 4000 rpm for 10 min, washed three times with sterile PBS, flash-frozen in liquid nitrogen, and stored at -80°C. Three biological replicates were established, along with a DMSO solvent control group (three replicates).

[0049] 1.5.2 RNA Extraction and Quality Detection

[0050] Total RNA was extracted using TRIGene Reagent reagent, and genomic DNA was removed using DNase I. RNA purity (OD) was determined using NanoDrop 2000. 260 / 280 =1.8-2.2, OD 260 / 230 RNA integrity was measured using the Agilent 2100 Bioanalyzer (RIN ≥ 6.5, 28S:18S ≥ 1.0). The total RNA amount per sample was ≥ 10 μg.

[0051] 1.5.3 Construction and Sequencing of cDNA Libraries

[0052] Strand-specific libraries were constructed using the TruSeq™ Stranded RNA Sample Prep Kit. Starting with 5 μg of total RNA, rRNA was removed using the Ribo-Zero Magnetic Kit. After fragmentation, cDNA synthesis, end repair, A-tail addition, and adapter ligation were performed. Fragments of 200-300 bp were selected by 2% low-melting-point agarose gel electrophoresis, amplified by 15 cycles of Phusion® DNA polymerase, and quantified using a TBS380 Picogreen. The libraries were then subjected to paired-end sequencing (150 bp × 2) on an Illumina NovaSeq 6000 platform.

[0053] 1.5.4 Data Quality Control and Comparison

[0054] Raw data were quality controlled using Trimmomatic (v0.36) (parameters: SLIDINGWINDOW:4:15, MINLEN:75) to remove adapter sequences and low-quality reads. Clean reads were then aligned to the Clostridium perfringens CVCC2030 reference genome (GenBank: NZ_CP028682.1) using Rockhopper (v2.0.3) in directional mode.

[0055] 1.5.5 Differentially expressed gene analysis

[0056] Gene expression levels were calculated using the RPKM method, and differential expression analysis was performed using edgeR (v3.28.1). Screening criteria: |log2FC|>1 and FDR<0.05. GO function enrichment was performed using Goatools (v0.6.5), and KEGG pathway enrichment was performed using KOBAS (v3.0). Bonferroni correction p<0.05 was considered significant enrichment.

[0057] 1.5.6 Protein-protein interaction network analysis

[0058] A differentially expressed gene PPI network was constructed based on the STRING database (v11.0) and visualized using Cytoscape (v3.7.2). The core hub genes were identified using the CytoHubba plugin's MCC algorithm, with a weight threshold of 0.10 and a minimum module size of 30 genes.

[0059] 1.6 Experimental Grouping and Treatment

[0060] One hundred and forty-four one-day-old male broiler chickens were acclimatized for 13 days. On day 14, the chickens were randomly divided into six groups: a blank control group (Con group), a model group (Mod group), a bromide group (BMD group), a low-dose resveratrol group (LRes group), a medium-dose resveratrol group (MRes group), and a high-dose resveratrol group (HRes group). Each group had three replicates, with eight chickens per replicate. The Con group was administered 1 mL of sterile PBS by gavage, while the other groups were administered 1 × 10⁻⁶ PBS by gavage. 5 1 mL of a solution of sporulated oocysts of *Eimeria tenella* per mL was administered. From day 17 to 23, the Con group received 1 mL of sterile PBS orally daily, while the other groups received a solution of 1 × 10⁻⁶ PBS daily. 9 1 mL of Clostridium perfringens bacterial suspension at CFU / mL was administered. From day 24 to 28, the BMD group was treated with 0.2 g of clostridial per liter of drinking water, while the LRes, MRes, and HRes groups were treated with 125 mg, 250 mg, and 500 mg of resveratrol per kilogram of feed, respectively. The remaining groups were fed a basal diet. The formal trial period was 15 days.

[0061] 1.7 Feeding and Management

[0062] All broilers were housed in well-ventilated rooms, with 23 hours of light and 1 hour of darkness per day. The room temperature was maintained at 33°C for the first week, then decreased by 1°C daily until reaching 26°C. From the start to the end of the experiment, the broilers had free access to food and water. They were also vaccinated according to a standard immunization schedule. The composition and nutritional levels of the basal diet are shown in Table 1.

[0063]

[0064] Note: 1. The premix provides the following per kilogram of feed: VA 10000 IU, VD 5200 IU, VE 40 IU, VK 8 IU, VB 15 IU, VB 28 IU, VB 6 12 IU, VB 12 2 IU, Biotin 0.2 mg, Niacin 45 mg, Folic Acid 0.8 mg, Iron 100 mg, Copper 8 mg, Zinc 1 mg, Manganese 120 mg, Iodine 0.3 mg, Selenium 0.7 mg.

[0065] 1.8 Growth performance

[0066] On days 14 and 29, after fasting the chicks for 12 hours while ensuring normal water supply, the chicks in each replicate group were weighed on an empty stomach. Feed intake throughout the entire experimental period was recorded to calculate average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR).

[0067] 1.9 Intestinal lesion score

[0068] On day 29, three chicks were randomly selected from each replicate. The chicks were bled from the jugular vein and then euthanized by dislocation of the neck. After necropsy to check the intestinal bloating, the jejunum and ileum were separated, and the intestinal lesions were scored according to Table 2.

[0069]

[0070] 1.10 Histopathological observation of jejunum

[0071] Well-fixed jejunal tissue samples were selected, stained with hematoxylin and eosin (HE), and prepared into pathological sections. Subsequently, the pathological changes of the jejunal tissue were observed using an Olympus BX35 biological microscope, and relevant images were acquired.

[0072] 1.11 Measurement of intestinal permeability indicators

[0073] This experiment determined intestinal permeability according to the method reported in the reference. The specific procedures were as follows: Two hours before sampling, two broiler chickens were randomly selected from each replicate group. Based on the chickens' body weight, 1 mL of FITC-d at a concentration of 4.17 mg / mL was administered orally per kilogram of body weight. After collecting blood from the wing vein, the chickens were euthanized by cervical dislocation. The collected blood samples were centrifuged at 3000 × g for 15 min at 4°C to obtain serum samples. Simultaneously, standard curves were plotted using different concentrations of FITC-d solution. The serum samples were then placed in a multi-mode microplate reader for fluorescence measurement and recording under conditions of excitation wavelength 485 nm and emission wavelength 528 nm. Finally, the concentration of FITC-d in the serum samples was calculated based on the plotted standard curves.

[0074] 1.12 Expression of jejunal barrier-related genes

[0075] First, total RNA was extracted from the jejunum using TRIGene Reagent. After extraction, the absorbance of the sample at 260 nm and 280 nm wavelengths was measured using a Multiskan SkyHigh automated microplate reader to determine the purity and concentration of RNA. Subsequently, the RNA was reverse transcribed into cDNA using StarScript III one-tube degenomic reverse transcription premix. Next, following the instructions, the cDNA, primers, and 2×RealStar Fast SYBR qPCR Mix were mixed, and the mixture was then placed in a Roche LightCycler 480 II real-time quantitative PCR instrument for quantitative PCR experiments. To ensure the reliability of the experimental results, each sample was tested in triplicate. β-actin was selected as the internal control gene, and the relative expression levels of each gene were calculated using the 2-ΔΔCt method. The primers used in the experiment were manufactured by Beijing Qingke Biotechnology Co., Ltd., and the specific gene primer sequences are shown in Table 3.

[0076]

[0077] 1.13 Determination of antioxidant performance indicators

[0078] Total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) in serum were determined using a kit. The detection methods were based on the instructions of the kit (Nanjing Jiancheng Bioengineering Co., Ltd.).

[0079] 1.14 Measurement of immune function indicators

[0080] Immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM), and calprotectin in jejunal tissue were measured using an ELISA kit. The detection methods were performed according to the kit instructions (Jiangsu Jingmei Biotechnology Co., Ltd.).

[0081] 1.15 Data Statistics and Analysis

[0082] First, the experimental data were organized using Excel (WPS Office 2024), then graphed using GraphPadPrism 9.0 software, and finally one-way ANOVA was performed using SPSS 27.0 statistical software. Duncan's method was used to perform multiple comparisons and mark significant differences. P < 0.05 indicated significant differences.

[0083] 2. Test Results

[0084] 2.1 In vitro inhibitory effect of resveratrol on Clostridium perfringens

[0085] 2.1.1 Minimum Inhibitory Concentration

[0086] As shown in Table 4, the MIC of resveratrol against Clostridium perfringens CVCC2030 was 0.16 mg / mL, which was comparable to the antibacterial effect of the positive control drug bacitracin (MIC=0.16 mg / mL). The DMSO solvent control group showed no antibacterial activity at a final concentration ≤1%, confirming that the antibacterial effect of resveratrol was independent of the solvent effect.

[0087]

[0088] Note: "-" indicates no bacterial growth, "+" indicates bacterial growth; MIC is the lowest concentration that is "+".

[0089] 2.1.2 Sliding motion inhibition

[0090] Compared with the Con group, as the resveratrol concentration increased from 0.08 mg / mL to 0.32 mg / mL, the diameter of the bacterial sliding zone decreased stepwise, to 15.2 mm, 10.5 mm, and complete inhibition, respectively.

[0091] 2.1.3 Inhibition of biofilm formation

[0092] Compared with the Con group, within the tested concentration range (0.02-5.00 mg / mL), the biofilm inhibition rate increased with increasing resveratrol concentration, exhibiting a typical dose-response relationship. When the concentration was ≥0.31 mg / mL, the inhibition rate exceeded 50%; when the concentration was ≥1.25 mg / mL, the inhibition rate stabilized at over 90%, approaching complete inhibition.

[0093] 2.2 Transcriptome regulatory mechanism of resveratrol on Clostridium perfringens

[0094] 2.2.1 Sequencing data quality assessment and differentially expressed gene screening

[0095] After quality control, the raw transcriptome sequencing data from the six samples (three replicates from the resveratrol treatment group and three replicates from the DMSO control group) yielded effective data ranging from 6.63 to 7.09 Gb / sample, with a Q30 base percentage >95.8% and an effective read retention rate >97%. PCA analysis showed clear separation between the two groups of samples, with good clustering of biological replicates, indicating high data reliability.

[0096] Using |log2FC|>1 and FDR<0.05 as screening criteria, a total of 512 differentially expressed genes (DEGs) were screened in the resveratrol treatment group (MIC concentration, 0.16 mg / mL) compared with the control group, of which 252 genes were significantly upregulated and 260 genes were significantly downregulated.

[0097] 2.2.2 Functional enrichment analysis of differentially expressed genes

[0098] 2.2.2.1 GO Functional Enrichment Analysis

[0099] GO analysis of 512 DEGs revealed that downregulated genes were significantly enriched in functions such as membrane transport, ion channels, and cellular structure. The most significant enrichment was in transmembrane transport protein activity (GO: 0022857, P = 1.02 × 10⁻⁶). -5 (Involving 20 genes), followed by ion channel activity (GO:0005216, P=3.70×10). -4 ) and ATPase-coupled ion transmembrane transport activity (GO: 0042625, P = 9.28 × 10) -4 Cellular component analysis showed that 41 downregulated genes were enriched in the cell membrane (GO: 0005886, P = 5.12 × 10⁻⁶). -5 (48.2%), indicating that resveratrol may disrupt membrane integrity. Nucleotide biosynthesis-related pathways (GO:0009156, P=1.59×10⁻⁶) -3 The activity was also inhibited, suggesting that energy metabolism was impaired.

[0100] The upregulated genes were mainly enriched in catabolism and stress responses, such as organic acid catabolism (GO:0016054, P=4.23×10). -3 ) and stress response (GO:0006950, P=2.33×10) -2 This reflects the compensatory stress response of bacteria.

[0101] 2.2.2.2 KEGG Functional Enrichment Analysis

[0102] A total of 83 enrichment pathways were identified, of which 9 were significantly enriched (P<0.05). The most significantly enriched pathway was teichoic acid biosynthesis (ko00552, P=3.70×10⁻⁶). -5 Of the 12 DEGs, 11 were downregulated, including key cell wall virulence structural genes such as tagO and tarI; the peptidoglycan biosynthesis pathway was also generally downregulated (8 downregulated vs. 1 upregulated). All 7 genes in the oxidative phosphorylation pathway were downregulated, and all 5 carbohydrate transport genes in the phosphotransferase system (PTS) were downregulated (P=0.021), indicating impaired energy metabolism. The ABC transporter pathway showed bidirectional regulation (15 upregulated, 14 downregulated), reflecting a reprogramming of bacterial nutrient uptake strategies. Furthermore, the two-component system regulatory genes virR (virulence regulation) and cheY (chemotaxis) were downregulated, while csgD (biofilm) was upregulated, possibly as a stress compensation response. All 9 genes in the ribosome pathway were downregulated, blocking protein synthesis.

[0103] 2.2.3 Identification of core antibacterial targets

[0104] Based on KEGG enrichment analysis, 49 DEGs were extracted from the cell wall synthesis (ko00552, ko00550), energy metabolism (ko00190), and two-component system (ko02020) pathways to construct a PPI subnetwork. This network contains 9 interacting nodes and 13 edges, forming a functional module with cell wall synthesis as the core.

[0105] The CytoHubba MCC algorithm was used to identify core hub genes. murA (UDP-N-acetylglucosamine 1-carboxylvinyltransferase, MCC=5.0) catalyzes the first irreversible step in peptidoglycan synthesis and is the first-ranked core gene; ddl (D-alanine-D-alanine ligase, MCC=4.0) is responsible for peptidoglycan precursor synthesis; bceA (bacitracin ABC transporter ATP-binding protein, MCC=4.0) is coupled to the two-component system; and uppP (undecyl isopentenyl bisphosphatase, MCC=3.0) participates in teichoic acid synthesis. The network showed that murA, ddl, and uppP form tight interaction clusters with multiple homologous genes, indicating that resveratrol disrupts cell wall integrity by synergistically inhibiting peptidoglycan and teichoic acid synthesis.

[0106]

[0107] 2.2.4 Transcriptome-Phenomenon Association Analysis

[0108] Transcriptome data were highly consistent with the in vitro antibacterial phenotype: a MIC value of 0.16 mg / mL corresponded to widespread inhibition of cell wall synthesis (downregulation of 11 genes including tagO and tarI), peptidoglycan synthesis (downregulation of 8 genes including murA and murC), and ribosome (downregulation of all 9 genes) pathways. Structural defects and protein synthesis blockade synergistically led to bacterial death. Complete inhibition of sliding motion (≥0.32 mg / mL) was consistent with the downregulation of cheY chemotactic regulatory genes in the two-component system, indicating that resveratrol interferes with motor signal transduction. Biofilm dose-dependent inhibition (≥1.25 mg / mL, inhibition rate >90%) exhibited a unique pattern: upregulation of the csgD gene was a bacterial stress compensation response, but impaired cell wall synthesis and downregulation of the PTS system and oxidative phosphorylation led to energy depletion, causing biofilm assembly failure.

[0109] 2.2.5 Molecular mechanism of resveratrol's antibacterial effect

[0110] Based on the above results, resveratrol inhibits Clostridium perfringens through a four-pronged mechanism of "structural synthesis-energy metabolism-protein synthesis-motor regulation": (1) inhibition of cell wall synthesis: downregulation of genes such as tagO and murA disrupts the synthesis of mitochondrial acid and peptidoglycan; (2) energy metabolism disorder: downregulation of genes related to oxidative phosphorylation and the PTS system blocks ATP generation and carbon source utilization; (3) protein synthesis blockage: complete downregulation of ribosome 9 gene stops protein synthesis; (4) loss of motility: downregulation of cheY inhibits chemotaxis. Biofilm stress failure stems from the inability of upregulation of csgD to overcome the dual defects of structure and energy. The synergistic effect of multiple targets leads to bacterial death and loss of virulence.

[0111] 2.3 Effects of resveratrol on growth performance of broilers infected with necrotic enteritis

[0112] like Figure 1 As shown, compared with the Con group, the Mod group showed significant differences in mean daily feed intake, mean daily weight gain, and feed conversion ratio. Compared with the Mod group, the BMD group and the MRes group also showed significant differences in mean daily feed intake, mean daily weight gain, and feed conversion ratio.

[0113] 2.4 Effect of resveratrol on intestinal lesion scores in broilers infected with necrotic enteritis

[0114] like Figure 2 As shown, compared with the Con group, all groups exhibited varying degrees of intestinal lesions in the duodenum, jejunum, and ileum. The Mod group showed the most severe intestinal lesions, with numerous hemorrhagic plaques. The BMD group showed the mildest intestinal lesions. With increasing resveratrol concentration, the intestinal lesions in the resveratrol-treated groups gradually lessened. The MRes group showed the mildest lesions, with duodenal, jejunal, and ileal scores decreasing by 58.3%, 62.5%, and 55.6% respectively compared to the Mod group, demonstrating superior efficacy compared to the BMD group. This indicates that resveratrol can alleviate the symptoms of necrotic enteritis in chickens.

[0115] 2.5 Effects of resveratrol on intestinal morphology of broiler chickens infected with necrotic enteritis

[0116] like Figure 3 As shown, the Con group showed no obvious intestinal damage, villus breakage, or dissolution; the Mod group exhibited severe intestinal integrity disruption, with significant villus breakage, dissolution, and detachment. Compared to the Mod group, villus breakage, dissolution, and damage gradually improved with increasing resveratrol concentration. The villus height / crypt depth ratio (V / C) in the MRes group was 42.3% higher than that in the Mod group, indicating restored mucosal epithelial integrity. Intestinal damage and villus breakage were less severe in the BMD group compared to the resveratrol group.

[0117] 2.6 Effects of resveratrol on intestinal permeability in broilers infected with necrotic enteritis

[0118] like Figure 4 As shown, compared with the Con group, the serum FITC-d concentration in the Mod group was significantly increased (P<0.05). After drug treatment, compared with the Mod group, the serum FITC-d concentrations in the LRes, MRes, HRes, and BMD groups were all significantly decreased (P<0.05). The FITC-d concentration in the MRes group was 35.2% lower than that in the Mod group and significantly lower than that in the BMD group (P<0.05), indicating a significant improvement in intestinal barrier function.

[0119] 2.7 Effects of resveratrol on the relative expression levels of jejunal barrier-related genes in broilers infected with necrotizing enteritis

[0120] like Figure 5 As shown, compared with the Con group, the mRNA expression levels of Occludin, ZO-1, and MUC-2 in the Mod group were significantly decreased (P<0.05). Compared with the Mod group, the mRNA expression levels of Occludin and MUC-2 in the LRes, MRes, HRes, and BMD groups were significantly increased (P<0.05), and the expression of ZO-1 in the MRes group also showed an increasing trend (P<0.05). The expression levels of Occludin and MUC-2 in the MRes group recovered to more than 85% of the levels in the Con group.

[0121] 2.8 Effects of resveratrol on antioxidant properties of broilers infected with necrotizing enteritis

[0122] like Figure 6 As shown, antioxidant indicators can reflect the body's ability to withstand oxidative stress, and these indicators can be used to analyze the antioxidant level of broilers. Compared with the Con group, the activities of T-AOC, CAT, and SOD in the serum of the Mod group were significantly decreased (P<0.05), while the content of MDA in the serum was significantly increased (P<0.05). After drug treatment, compared with the Mod group, the activities of T-AOC, CAT, and SOD in the serum of the HRes group and BMD group were significantly increased (P<0.05), while the content of MDA in the serum of the MRes group, HRes group, and BMD group was significantly decreased (P<0.05). The SOD activity in the HRes group was increased by 31.2% and the MDA was decreased by 28.6% compared with the Mod group, showing a dose-dependent increase in antioxidant effect.

[0123] 2.9 Regulatory effect of resveratrol on intestinal inflammation in broilers infected with necrotic enteritis

[0124] like Figure 7As shown, detecting pro-inflammatory and anti-inflammatory factors can effectively reflect the degree of inflammation in broilers and can also be used to evaluate the anti-inflammatory efficacy of drugs. Compared with the Con group, the concentrations of pro-inflammatory factors IL-1β, IL-6, and TNF-α in the jejunal tissue of the Mod group were significantly increased (P<0.05), while the concentration of anti-inflammatory factor IL-10 was significantly decreased (P<0.05), indicating that the broiler body was in a state of "pro-inflammatory dominance". After drug treatment, compared with the Mod group, the concentrations of pro-inflammatory factors IL-1β, IL-6, and TNF-α in the jejunal tissue of the LRes, MRes, HRes, and BMD groups were significantly decreased (P<0.05), while the concentration of anti-inflammatory factor IL-10 was significantly increased (P<0.05). In the MRes group, IL-6 decreased by 42.3%, and IL-10 increased by 58.7%, restoring the level of inflammatory factors to near that of the Con group, achieving a "suppressive-anti-inflammatory" balance.

[0125] 2.10 The regulatory effect of resveratrol on mucosal immunity in broilers infected with necrotizing enteritis

[0126] like Figure 8 As shown, compared with the Con group, the concentrations of calprotectin, IgA, IgM, and IgY in the jejunal tissue of the Mod group were significantly increased (P<0.05), reflecting mucosal inflammatory damage and compensatory immune activation. After drug treatment, compared with the Mod group, the calprotectin levels in the LRes, MRes, HRes, and BMD groups were significantly decreased (P<0.05), indicating reduced inflammatory damage; the concentrations of IgA, IgM, and IgY were significantly downregulated compared with the Mod group (P<0.05), but still higher than those in the Con group, maintaining at normal immune response levels, suggesting that resveratrol avoids tissue damage caused by excessive immune activation.

[0127] In summary, resveratrol exhibits good therapeutic effects on necrotizing enteritis in chickens, improving the health of infected broilers through a dual mechanism of direct antibacterial action and intestinal repair. Specifically, in vitro, resveratrol directly inhibits Clostridium perfringens by downregulating the expression of genes related to cell wall synthesis (murA, ddl, uppP), energy metabolism (oxidative phosphorylation, PTS system), and virulence regulation (virR, cheY), thereby disrupting bacterial structural integrity and blocking its energy supply. In vivo, resveratrol significantly upregulates the gene expression of intestinal tight junction proteins Occludin, ZO-1, and mucin MUC-2, repairing the intestinal barrier structure, reducing intestinal permeability, and decreasing the invasion of harmful substances. Simultaneously, resveratrol balances the inflammatory response and regulates mucosal immunoglobulin levels by inhibiting pro-inflammatory factors IL-1β, IL-6, and TNF-α while promoting the expression of the anti-inflammatory factor IL-10, thereby enhancing the body's immune function and alleviating the decline in growth performance. Furthermore, resveratrol significantly increased the activity of serum antioxidant enzymes (SOD, CAT), reduced MDA content, effectively alleviated oxidative stress, and further promoted the recovery of growth performance. Among them, the medium dose of 250 mg / kg feed showed the most significant therapeutic effect, and the overall effect was better than or equivalent to the antibiotic control group.

Claims

1. A single monomer of traditional Chinese medicine, characterized in that, It is composed of resveratrol.

2. An application of the traditional Chinese medicine monomer according to claim 1, characterized in that, The resveratrol dosage is 125-500 mg per kilogram of feed.

3. The application of the traditional Chinese medicine monomer according to claim 2, characterized in that, The resveratrol dosage is 250 mg per kilogram of feed.

4. The application of a traditional Chinese medicine monomer according to claim 1 in the preparation of a drug for treating necrotizing enteritis in chickens.

5. The application of the traditional Chinese medicine monomer according to claim 4 in the preparation of a drug for treating necrotizing enteritis in chickens, characterized in that, The applications include their use in the preparation of drugs that enhance the immune function of broilers infected with necrotizing enteritis.

6. The application of the traditional Chinese medicine monomer according to claim 4 in the preparation of a drug for treating necrotizing enteritis in chickens, characterized in that, The applications include their use in the preparation of drugs to improve the growth performance of broilers infected with necrotizing enteritis.

7. The application of the traditional Chinese medicine monomer according to claim 4 in the preparation of a drug for treating necrotizing enteritis in chickens, characterized in that, The applications include their use in the preparation of drugs for repairing the intestinal barrier in broilers infected with necrotizing enterocolitis.

8. The application of the traditional Chinese medicine monomer according to claim 4 in the preparation of a drug for treating necrotizing enteritis in chickens, characterized in that, The applications include their use in the preparation of drugs that enhance the antioxidant properties of broilers infected with necrotizing enteritis.

9. The application of the traditional Chinese medicine monomer according to claim 4 in the preparation of a drug for treating necrotizing enteritis in chickens, characterized in that, The application includes its use in the preparation of drugs that directly inhibit the proliferation of Clostridium perfringens.

10. The application of the traditional Chinese medicine monomer according to claim 4 in the preparation of a drug for treating necrotizing enteritis in chickens, characterized in that, The necrotizing enteritis in chickens was caused by a mixed infection of Eimeria giantiformis and Clostridium perfringens type A.

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