Amino acid peptide for preventing or treating laminitis and use thereof

Abstract

This invention relates to the field of pharmaceutical technology and provides an amino acid peptide for the prevention or treatment of laminitis in horses and its applications. By applying an amino acid peptide with the amino acid sequence shown in SEQ ID NO.1 to the prevention and treatment of laminitis, this invention achieves multi-dimensional and multi-mechanism disease intervention effects. At the preventive level, this amino acid peptide can effectively improve the clinical abnormalities that horses may exhibit due to laminitis, while helping to restore the balance of the intestinal microbiota, providing protection against the risk of laminitis in horses. At the therapeutic level, it can significantly reduce lameness and diarrhea symptoms in horses, alleviate pathological damage such as hoof bleeding and edema, promote the repair of hoof lobule tissue structure, and gradually restore normal hoof physiological function. The entire intervention process is safe, does not cause abnormal fluctuations in body temperature in horses, and the injection administration method has not triggered any related adverse reactions, providing a novel and highly effective targeted treatment scheme for the prevention and treatment of laminitis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to an amino acid peptide for the prevention or treatment of lavillary dermatitis and its application. Background Technology

[0002] Laminitis is one of the most common and serious hoof diseases in equines. Its pathological characteristics include aseptic inflammation, hemorrhage, and degradation of the hoof laminae, ultimately leading to hoof bone displacement or sinking. Clinical manifestations include severe pain and lameness, and in severe cases, disuse or death. This disease not only causes immense suffering for animals but also results in significant economic losses.

[0003] Currently, clinical treatment strategies for laminitis mainly focus on the following aspects: (1) Symptomatic supportive treatment: This treatment aims to relieve pain and suppress inflammation. The core is the use of nonsteroidal anti-inflammatory drugs (NSAIDs), such as flunixin meglumine. However, these drugs only treat the symptoms and not the root cause. Long-term use can easily cause serious side effects such as gastrointestinal ulcers and kidney damage, and the effect on severe cases is limited. (2) Physical therapy: such as hoof ice bath (cold therapy), which aims to reduce inflammatory response and pain. However, this method can only be used as an auxiliary means and cannot reverse the pathological damage of the laminar layer. (3) Improvement of microcirculation and anti-matrix metalloproteinase (MMP) treatment: Studies have shown that the development of laminitis is closely related to hoof microcirculation disorders and abnormally increased activity of MMPs (such as MMP-2 and MMP-9), which leads to degradation of the basement membrane and extracellular matrix of the laminar layer. However, there are currently no specific drugs on the market that target this. (4) Treatment targeting inducing factors: such as preventing carbohydrate overload through dietary management, but this method is only for prevention and is ineffective for animals that have already developed the disease.

[0004] In recent years, with in-depth research, the "gut-hoof axis" theory has gradually gained acceptance. This theory posits that gastrointestinal dysfunction (such as oligofructose-induced gut microbiota dysbiosis) is a key initiating factor in laminitis. Gut microbiota dysbiosis can lead to the production and entry of harmful metabolites such as endotoxins (LPS), histamine, and lactic acid into the circulatory system, triggering a systemic inflammatory response and ultimately activating micromineral protein (MMP) in the hoof and lamellar layer, causing tissue damage. Therefore, a dual-mechanism therapy that simultaneously targets gut microbiota regulation and inhibits hoof and lamellar MMP activity is considered the most promising new strategy for treating laminitis. However, there are significant gaps in current technology: existing drugs (such as NSAIDs) can only relieve symptoms individually and cannot simultaneously target the two core pathological links of the disease, namely "gut origin" and "hoof injury," resulting in limited efficacy and significant side effects. Existing microbial preparations (such as probiotics) can regulate the gut microbiota, but their onset of action is slow, their effects are unstable, and they cannot directly inhibit hoof MMP activity, making them ineffective in treating acute flare-ups of laminitis. Currently, there are no amino acid peptide-based drugs or methods on the market that can provide comprehensive treatment for lavage by regulating the "gut flora-MMP axis".

[0005] In summary, there is an urgent need in this field to develop a drug with a novel mechanism of action that simultaneously meets the following requirements: (1) it can rapidly correct intestinal flora imbalance and reduce the production of harmful metabolites; (2) it can effectively inhibit systemic inflammatory responses; (3) it can strongly downregulate the activity of MMPs in the lamellar layer, preventing structural damage to the lamellar layer from the source; and (4) it has good safety profiles, avoiding the common toxic side effects of existing drugs. Therefore, this invention proposes an amino acid peptide for the prevention or treatment of lamellar dermatitis and its application. Summary of the Invention

[0006] The purpose of this invention is to provide an amino acid peptide for the prevention or treatment of lavage and its application, thereby addressing the problems raised in the background art.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] An amino acid peptide, the amino acid sequence of which is shown in SEQ ID NO.1, namely LKAMDPTPPLWIKTQ.

[0009] Furthermore, the amino acid peptide can regulate the intestinal microbiota of horses, including at least one of the following effects: increasing the relative abundance of Akkermania, increasing the relative abundance of Bacteroidetes, increasing the relative abundance of Trichophyceae, decreasing the relative abundance of Streptococcus, and decreasing the relative abundance of Firmicutes.

[0010] The use of an amino acid peptide according to the above description in the preparation of a medicament for the prevention or treatment of laminitis.

[0011] Furthermore, the prevention or treatment includes improving at least one of the following clinical parameters: increasing fecal pH, reducing Obel claudication score, and improving diarrhea score.

[0012] Furthermore, the prevention or treatment includes reducing the level of at least one of the following substances in the serum: endotoxin, 5-HT, IL-17A, IL-10, MMP-1, MMP-2, MMP-9.

[0013] A pharmaceutical composition for the prevention of lavillary dermatitis comprises the aforementioned amino acid peptides and sterile saline.

[0014] Furthermore, the drug was administered via intravenous injection into the jugular vein at the following time points: 48 hours before gavage, 24 hours before gavage, and on the day of gavage, with corresponding injection volumes of 150 mL, 150 mL, and 200 mL, respectively.

[0015] A pharmaceutical composition for treating lavillary dermatitis comprises the aforementioned amino acid peptide and sterile saline.

[0016] Furthermore, the medication was administered via jugular vein injection. The first injection was given when the horse exhibited symptoms of laminitis, such as fever, diarrhea, or an Obel lameness score >1. A second injection was given 12 hours after the first injection, with each injection volume being 250 mL.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] This invention utilizes an amino acid peptide with the amino acid sequence shown in SEQ ID NO.1 to prevent and treat laminitis in horses, achieving multi-dimensional and multi-mechanism disease intervention effects. At the preventative level, this amino acid peptide effectively improves the clinical abnormalities that horses may exhibit due to laminitis, while also helping to restore the balance of the intestinal microbiota, providing protection against the risk of laminitis. At the therapeutic level, it significantly reduces lameness and diarrhea symptoms in horses, alleviates pathological damage such as hoof bleeding and edema, promotes the repair of hoof lobule tissue structure, and gradually restores normal hoof physiological function. The entire intervention process is safe, does not cause abnormal fluctuations in body temperature in horses, and the injection administration method did not induce any related adverse reactions. Furthermore, this amino acid peptide overcomes the limitations of traditional single anti-inflammatory treatments. Through the synergistic effects of multiple mechanisms, including regulating intestinal flora imbalance, inhibiting the release of inflammatory mediators, improving hoof blood circulation, and repairing damaged tissue, it not only solves the clinical problem of limited and ineffective treatment methods for laminitis but also effectively addresses the recurrent lameness, pain, and local and systemic inflammation caused by the disease, providing a novel and highly effective targeted approach for the treatment of laminitis in the veterinary field. Attached Figure Description

[0019] Figure 1 For the purpose of studying the timeline overview diagram.

[0020] Figure 2 Comparison of clinical parameters of horses in different groups; where A is rectal temperature change; B is fecal pH value; C is lameness score; D is diarrhea score (d1-d4: fecal morphology diagram of control group, laminitis group, prevention group and treatment group).

[0021] Figure 3 The results of local hoof examinations are shown (from left to right: control group, laminitis group, prevention group, and treatment group); where A is Doppler ultrasound examination of the medial digital artery; B is hoof anatomy; C is H&E staining of hoof leaflet tissue; and D is PAS staining of hoof leaflet tissue.

[0022] Figure 4 The composition of fecal microbial communities in different groups is shown below; where A is the phylum-level microbial community structure; B is the genus-level microbial community structure; C is the phylogenetic clade diagram generated by LEfSe analysis (showing the taxonomic unit relationships at the phylum, class, order, family, genus, and species levels); and D is a Venn diagram showing the number of core bacterial genera in the fecal samples.

[0023] Figure 5 Pearson correlation analysis was performed on clinical parameters, serum indicators, and fecal microbiota composition for different groups.

[0024] Figure 6 Analysis of fecal microbial community composition for different groups; where A is the sparse curve; B is the Chao1 index; C is the Goods coverage; D is the number of observed species; E is the Pielou_e evenness index; F is the Shannon index; G is the Simpson index; H is principal coordinate analysis (PCoA); J is nonmetric multidimensional scaling analysis (NMDS); and K is the UPGMA clustering dendrogram.

[0025] Figure 7 Linear discriminant analysis (LDA) score plots (based on LEfSe analysis) show the iconic taxa of horse fecal microbiomes in different groups (LDA score > 3, and Wilcoxon signed-rank test significance p < 0.05).

[0026] Figure 8 PICRUSt2 functional pathway analysis for fecal microbiota in different groups. Detailed Implementation

[0027] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0028] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0029] This invention provides an amino acid peptide for the prevention or treatment of laminitis and its application. By clarifying the characteristics of the amino acid peptide, constructing a laminitis model, designing a scientific administration method, and conducting multi-dimensional detection and analysis, the preventive and therapeutic effects of the amino acid peptide on laminitis are verified. At the same time, the potential mechanism of its action is revealed, providing a new technical path for the treatment of laminitis.

[0030] (a) Basic characteristics of amino acid peptides;

[0031] The amino acid peptide involved in this invention is a synthetic peptide composed of 15 amino acids, with the amino acid sequence LKAMDPTPPLWIKTQ (as shown in SEQ ID NO.1). This amino acid peptide was first isolated from opossum serum in 1999. Early studies determined that this peptide could inhibit the effects of snake venom on opossums and mice. Subsequent studies found that this amino acid peptide and its shorter peptide derivatives have anti-inflammatory and immunomodulatory effects on chronic wound healing and other similar inflammatory conditions. However, regarding the specific effects of this amino acid peptide in the prevention and treatment of laminitis, no relevant research has been reported, indicating a research gap.

[0032] (II) Establishment of the zebufo var. zebufo model;

[0033] 1. Horse selection;

[0034] Twenty clinically healthy Mongolian horses (10 stallions and 10 mares; age 3 years; weight 285-335 kg) were selected from a single ranch's 2020 breeding herd (detailed horse information is provided in Tables S1 and S2). All horses were fattened under standardized nutrition and management conditions. Selection criteria included: an Obel lameness score of "0"; no pain response upon hoof clamp examination; no laminitis-related abnormalities on imaging (especially ruling out ventral rotation of the third phalanx); and good overall health confirmed through clinical observation during the acclimatization period and before induction. Pre-selection horses were subject to pre-existing conditions excluding: intermediate pituitary dysfunction (Cushin disease), equine metabolic syndrome (insulin resistance), history of antibiotic treatment, gastrointestinal diseases, and behavioral abnormalities. Additionally, a body condition score >3 and <7 was required, and mares were not pregnant or lactating.

[0035] Table S1. Breed, sex, and age of the horses used

[0036]

[0037] Table S2. Horses' physical condition scores and weight

[0038]

[0039] 2. Standardized feeding and management;

[0040] Considering the significant impact of diet on the equine gut microbiota, and to ensure a highly controllable experimental environment, this study implemented additional strict feeding and management measures, even though all horses were kept under the same feeding conditions and exercise program: after a 30-day isolation acclimatization period, all horses were fed a standardized hay ration (nutrient composition detailed in Table 1). The daily ration was precisely calculated at 2% of each horse's body weight (dry matter basis) and administered in two equal portions at 07:00 and 17:00. During the feeding period, horses had free access to water and hay, but any supplementary feeds were prohibited to avoid confounding the microbiota profile. This protocol met all the maintenance requirements of adult horses while ensuring consistent nutrient intake, thereby eliminating the interference of dietary variables on the experimental results.

[0041] Table 1. Chemical composition of horse feed

[0042]

[0043] 1 Dry matter; 2 Natural substances; 3 Crude protein; 4 Crude fat; 5 Neutral detergent fibers; 6 Acid detergent fibers; 7 calcium; 8 phosphorus.

[0044] 3. Fructooligosaccharide-induced modeling;

[0045] An experimental laminitis model in horses was established using a fructooligosaccharide (FOS) induction method. The specific procedure was as follows: After the horses adapted to the above-mentioned dietary and environmental conditions, the experimental groups (including the laminitis group, prevention group, and treatment group) were fed 1 g / kg body weight of FOS, added to their daily feed. This adaptation phase lasted for 3 days to allow the horses' gastrointestinal system to gradually adapt to FOS. After the adaptation phase, the experimental group horses received 10 g / kg body weight of FOS (pre-dissolved in 10 L of tap water) via a nasogastric tube to ensure accurate and uniform ingestion of the prescribed dose of FOS. Figure 1 Afterwards, the horses were returned to their original feeding environment and management conditions were restored. At the same time, the horses' condition after being administered fructooligosaccharides was closely observed to record and analyze any possible clinical symptoms.

[0046] (iii) Dosing regimens for amino acid peptides;

[0047] Twenty horses were divided into four groups: a control group (Healthy, n=5), a laminitis group (Laminitis, n=5), an amino acid peptide prevention group (Prophylactic, n=5), and an amino acid peptide treatment group (Treatment, n=5).

[0048] Both the prevention and treatment groups of horses were administered the medication via jugular intravenous injection to ensure rapid entry into the bloodstream and its effectiveness. Before administration, 4g of amino acid peptide was thoroughly mixed with 500mL of sterile physiological saline (0.9%) to prepare a homogeneous solution, ensuring complete drug dissolution. The administration strategies for the two groups were differentiated based on their intervention objectives: In the prevention group, each horse received three jugular intravenous injections to provide sufficient amino acid peptide protection before fructooligosaccharide-induced laminitis. Specific time points were: 48 hours before gavage (-48h), 24 hours before gavage (-24h), and on the day of gavage (0h), with corresponding injection volumes of 150mL, 150mL, and 200mL of amino acid peptide solution, respectively. In contrast, the horses in the treatment group received only two jugular intravenous injections to simulate an emergency treatment scenario following the onset of laminitis. The first injection was given when the horse showed symptoms of fever, diarrhea or laminitis (Obel>1), and the second injection was given 12 hours after the first injection. The volume of each injection was 250 mL of amino acid peptide solution.

[0049] This study included a prevention group, which, in addition to providing a comparison of intervention timing with the treatment group, focused on exploring the application potential of amino acid peptides in the prevention of laminitis. Considering that high-intensity exercise may increase the risk of laminitis in horses, this group design could provide experimental evidence for the preventative medication of sport horses before high-intensity training or competitions, thereby offering new intervention strategies for the prevention and control of laminitis.

[0050] (iv) Multi-dimensional detection and analysis methods;

[0051] 1. Clinical examination;

[0052] Throughout the study, clinical parameters (rectal temperature, fecal pH, lameness score, and diarrhea score) were continuously monitored. The methods for measuring each clinical parameter are as follows: Rectal temperature: Measured and recorded rectally using a digital thermometer (GIMA, Italy). Fecal pH: Fresh fecal samples collected from the rectum were directly measured using a calibrated pH meter (Baty, China). Lameness score: The Obel scoring system was used to grade the severity of laminitis (0-4 grades) based on gait abnormalities, weight-bearing willingness, and pain response. Diarrhea score: A 0-5 grading system was used, specifically: 0 (normal), 1 (soft, formed stool), 2 (pudding-like stool with form), 3 (pudding-like stool without form), 4 (watery stool with fragments), 5 (completely liquid stool).

[0053] Clinical assessments were performed every 6 hours after administration of fructooligosaccharides (FOS) (the same time point for the control group). Intergroup comparisons used disease-specific endpoint data: 36 hours after induction (peak period of pathological changes) for the laminitis group, and 72 hours (optimal time for efficacy assessment) for the control, prevention, and treatment groups. This time-stratified design considered both the differences in disease progression and the direct comparison of intervention effects between the treatment group, the laminitis group, and the control group. Monitoring results are shown in Tables S3-S6.

[0054] Table S3. Clinical assessment (rectal temperature) should be performed every 6 hours after administration of fructooligosaccharides.

[0055]

[0056] Table S4. Clinical assessment (fecal pH) should be performed every 6 hours after administration of fructooligosaccharides.

[0057]

[0058] Table S5. Clinical assessment (claudication score) should be performed every 6 hours after fructooligosaccharide administration.

[0059]

[0060] Table S6. Clinical assessment (diarrhea score) should be performed every 6 hours after administration of fructooligosaccharides.

[0061]

[0062] 2. Doppler ultrasound measurement;

[0063] A standardized Doppler ultrasound system (Hoyosun Technology, China) equipped with a 10MHz linear array probe was used to assess the hemodynamic parameters of the medial digital artery. Each horse underwent three consecutive measurements under constant environmental conditions (room temperature 20-22℃, resting heart rate), with the probe maintained at a 60° angle of incidence to ensure accurate flow velocity calculations. Each measurement included peak systolic velocity (PSV) and minimum end-diastolic velocity (EDV). To reduce variability, all measurements were performed by the same experienced operator according to a pre-defined anatomical landmark protocol, and the average of the three measurements was used for analysis.

[0064] 3. LPS concentration determination in serum;

[0065] After collecting blood samples, they were centrifuged at 14000×g for 30 minutes at 4°C. The supernatant was then transferred to sterile, pyrogen-free glass tubes. Endotoxin (LPS) concentrations were determined using a colorimetric endpoint method (test kit purchased from MLBIOBiotechnology Co., Ltd., Shanghai, China) according to the manufacturer's instructions. This method has a minimum detection threshold of 0.01 EU / mL, enabling accurate measurement of LPS concentrations.

[0066] 4. Detection of serum biomarker concentrations;

[0067] Blood samples were collected from each group of horses and centrifuged at 3000×g for 30 minutes at 4°C. Serum was collected to detect the concentrations of the following indicators: lactate, histamine, insulin, IL-17A, IL-10, serum amyloid A (SAA), p38-MAPK, serotonin (5-HT), thromboxane B2 (TXB2), and matrix metalloproteinases (MMP-1, MMP-2, MMP-9, MMP-14). All tests were performed strictly in accordance with the operating procedures of the test kit manufacturer (MLBIO Biotechnology Co. Ltd).

[0068] 5. Total protein concentration detection;

[0069] After collecting blood samples from each group of horses, the samples were centrifuged at 3000×g for 30 minutes at 4°C. The serum was then separated and the total protein concentration was determined using an IDEXX VetAutoread biochemical analyzer (IDEXX, USA).

[0070] 6. Euthanasia procedure;

[0071] To obtain hoof and laminar flow tissue samples for pathological evaluation, this study selected three horses with laminitis from each group for euthanasia to ensure sufficient sample size for accurate analysis of pathological changes. The entire procedure strictly followed scientifically validated standard procedures for equine euthanasia, balancing scientific rigor with humanitarian principles. The specific process was as follows: A specific ratio (1:5) of xylazine and ketamine (Fuli Pharmaceutical Co., Ltd., Lanzhou, China) was administered intravenously via the jugular vein at a rate of 0.5-1 mL / s. This combination rapidly induces unconsciousness and reduces pain in the horses. Once the horses were deeply sedated, sodium pentobarbital (Feiyue Pharmaceutical Co., Ltd., Heilongjiang, China) was immediately injected at a dose of 0.1 mL / kg to ensure rapid and painless death. The entire process was monitored by veterinary experts. Methods such as cardiac arrest, eye contact, and ear pricking were used to rigorously verify that the horses were completely unconscious and pain-free, ensuring the euthanasia procedure met humane standards before proceeding with subsequent tissue collection.

[0072] 7. Anatomical observation of the hoof;

[0073] A comprehensive assessment of the hoof's internal structure is conducted through hoof dissection to better understand the pathological changes and treatment outcomes of laminitis. This process requires extremely meticulous examination and analysis of the hoof's internal structures to ensure no potential pathological signs are missed, assessing its health and integrity. Particular attention is paid to various anatomical components, including the hoof folds and hoof walls, as these are among the most important anatomical parts of the hoof. These structures are carefully observed, searching for any signs that might indicate a pathological condition, such as fold lesions, inflammation, or degradation—typical pathological changes that can occur in laminitis. Furthermore, photographic documentation and detailed observation are used to capture and record any important findings or noteworthy features during the dissection process.

[0074] 8. Hematoxylin-eosin (H&E) and periodic acid Schiff staining (PAS);

[0075] To ensure sample quality and integrity, lamellar tissue surrounding the hoof lobes was rapidly collected within one hour of euthanasia and cut using a handsaw. To ensure consistency and comparability across all samples, the hoof lobes were precisely cut into 10×10×0.5 mm sections, covering the hoof lobes and lamellar tissue. These sections were then divided into 55 mm tissue cubes and fixed in 4% formaldehyde solution for 24–72 hours. This fixation time was consistent across all samples, ensuring optimal tissue preservation. After fixation, the fixed lamellar tissue cubes were paraffin-embedded using standard methods. This step protects the tissue structure and facilitates subsequent sectioning and staining. For consistency, special care was taken to obtain tissue cubes from the same location within the lamellar tissue of each horse. For assessment of lamellar lesions and morphological analysis, the paraffin-embedded sections were stained using H&E and PAS staining methods. After staining, the sections were examined meticulously using a high-resolution microscope, and high-quality, fully digital images were acquired using specialized image capture software. This was crucial for subsequent analysis and research.

[0076] 9. Microbiological assessment;

[0077] At the end of the study (36 hours after induction in the laminitis group; 72 hours in the control, prevention and treatment groups), fecal samples were collected from 20 horses (5 horses in each group) for bacterial DNA extraction and subsequent microbiological analysis.

[0078] DNA was extracted from horse manure samples using the cetyltrimethylammonium bromide (CTAB) method according to the manufacturer's instructions. This method is suitable for DNA recovery from trace samples and has been widely validated for DNA preparation from a variety of bacterial species. Blank control samples were treated with nuclease-free water. The extracted total DNA was dissolved in 50 μL of elution buffer and stored at -80°C until subsequent analysis.

[0079] To amplify the V3-V4 region of the bacterial 16S rDNA gene, a set of barcoded primers was used: 314F (5ʹ-CCTACGGGNGGCWGCAG-3ʹ, as shown in SEQ ID NO.2) and 805R (5ʹ-GACTACHVGGGTATCTAATCC-3ʹ, as shown in SEQ ID NO.3), with universal sequencing primer sequences ligated to the 5ʹ ends of both primers. The total volume of the PCR reaction system was 25 μL, containing 25 ng template DNA, 12.5 μL of PCR premix, and 2.5 μL of each primer, with the volume made up to PCR-grade water. The amplification conditions for the prokaryotic 16S fragment were: 98°C pre-denaturation for 30 seconds; 32 cycles (98°C denaturation for 10 seconds, 54°C annealing for 30 seconds, 72°C extension for 45 seconds); and a final extension at 72°C for 10 minutes. The amplified products were verified by 2% agarose gel electrophoresis. Throughout DNA extraction, ultrapure water was used as a negative control instead of the sample solution to rule out false positives. PCR products were purified using AMPure XT magnetic beads (Beckman Coulter Genomics, USA), quantified using Qubit (Invitrogen, USA), and then used to construct amplicon libraries. The size and concentration of library fragments were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and an Illumina library quantification kit (Kapa Biosciences, USA), respectively. Finally, sequencing was performed on an Illumina NovaSeq PE250 platform according to the manufacturer's standard procedures.

[0080] Paired-end reads of samples were distinguished by unique barcodes, and primer sequences were removed. Paired-end reads were merged using FLASH software, and the raw reads were quality controlled using fqtrim (v0.94) under preset filtering conditions to generate high-quality clean tags. After DADA2 deduplication, chimeric sequences were removed using Vsearch (v2.3.4), finally obtaining the feature table and sequence data. To maintain consistency, all sequences were randomized before α and β diversity analysis. Eigenvalues ​​were standardized based on the relative abundance of samples using the SILVA database classifier.

[0081] Multiple methods were employed to analyze the microbial structure of horse manure samples from different groups, including the number of observed species, Chao1 index, Shannon index, Simpson index, Pielou'e evenness index, principal coordinate analysis (PCoA), UPGMA clustering, and nonmetric multidimensional scaling (NMDS). Linear discriminant analysis (LEfSe) was used to identify bacterial communities with significant differences between groups. Furthermore, Pearson correlation analysis was conducted by LC-Bio Technology (https: / / www.omicstudio.cn / ) to explore the correlation between fecal microbiota and host parameters and serum concentrations. Finally, PICRUSt2 (a community phylogenetic study using unobserved state reconstruction) was used to analyze changes in bacterial function across different groups.

[0082] 10. Statistical analysis;

[0083] All statistical analyses in this study were performed using GraphPad Prism software (version 10.0.3, GraphPad Software, San Diego, California, USA). Data are presented as mean ± standard error (mean ± SEM). The Alpha diversity index was calculated using the Kruskal-Wallis test. Pearson correlation analysis was used to explore the association between microbial community composition and clinical parameters. A p-value < 0.05 was set as the statistical significance threshold, and the significance levels were labeled as follows: p < 0.05, p < 0.01, p < 0.001, ****p < 0.0001.

[0084] (v) Experimental results;

[0085] 1. The effects of amino acid peptide intervention on clinical parameters;

[0086] To evaluate the clinical efficacy of amino acid peptide intervention, rectal temperature, fecal pH, Obel score, and diarrhea score were selected as observation indicators. Figure 2 After 72 hours of fructooligosaccharide induction, compared with the control group, the laminitis group showed significantly increased rectal temperature (p=0.0085), increased lameness score (p=0.0056), and increased diarrhea score (p=0.0001), while the fecal pH value decreased (p<0.0001). After amino acid peptide intervention, the clinical parameters of the prevention and treatment groups were improved compared with the laminitis group: the fecal pH value of the prevention group (p=0.0089) and the treatment group (p=0.0072) significantly increased. Figure 2 (Medium B); Limping scores decreased significantly (p=0.0045 and p=0.0056, respectively) Figure 2(C) Diarrhea scores significantly improved (p=0.0001; p=0.0004), and stool consistency and color showed improvement (C). Figure 2 (D). Notably, amino acid peptide intervention did not have a significant effect on rectal temperature parameters (p=0.1035; p=0.0627). Figure 2 (A)

[0087] 2. Effects of amino acid peptide intervention on blood circulation, anatomical features, and hoof leaflet structure;

[0088] To assess the effects of amino acid peptides on hoof hemodynamics, the experiment used Doppler ultrasound of the medial digital artery (DFA) for detection. Figure 3 (A) Horses in the control group exhibited a high-low resistance blood flow waveform, while horses in the laminitis group showed significantly reduced peak systolic velocity (PSV: control group 42.0±0.3cm / s, laminitis group 14.7±0.2cm / s, p<0.0001) and end-diastolic velocity (EDV: control group 19.87±0.03cm / s, laminitis group 6.99±0.02cm / s, p<0.0001), indicating impaired vascular perfusion. After amino acid peptide intervention, both the prevention group (PSV: 33.79±0.02cm / s; EDV: 10.89±0.02cm / s, p<0.0001) and the treatment group (PSV: 38.38±0.02cm / s; EDV: 18.36±0.02cm / s, p<0.0001) showed improved arterial perfusion, with the hemodynamic parameters of the treatment group being close to those of the control group.

[0089] Macroscopic anatomy of the hoof shows ( Figure 3 In the treatment group (B group), significant bleeding and edema were observed compared to the control group. Amino acid peptide intervention alleviated these pathological changes, with bleeding and swelling significantly reduced, particularly in the treatment group.

[0090] Histopathological evaluation (H&E and PAS staining) showed ( Figure 3 (C and D) The affected horses exhibited typical pathological features of laminitis, including epidermal-dermal lobule separation, neutrophil infiltration, and hoof wall structural destruction. After treatment with amino acid peptides, both the prevention and treatment groups showed reduced inflammatory cell infiltration, improved lobule structure arrangement, and partial restoration of epidermal continuity. The treatment group showed near-complete histological repair, with structural features highly similar to the control tissue.

[0091] 3. Effects of amino acid peptide intervention on serum inflammatory markers, MMPs, and metabolites;

[0092] Table 2 shows that serum inflammatory markers (LPS [p=0.0177], IL-17A [p=0.0032], IL-10 [p<0.0001]), matrix metalloproteinases (MMP-1 [p=0.0026], MMP-2 [p=0.0280], MMP-9 [p=0.0465]), and metabolic mediators (lactic acid [p=0.0001], histamine [p=0.0246], insulin [p=0.0081], 5-HT [p=0.0009]) in horses with laminitis were significantly higher than those in the control group. Following amino acid peptide intervention, the prevention and treatment groups exhibited differentiated effects: the prevention group primarily showed significant reductions in IL-17A (p=0.0162), MMP-9 (p=0.0141), and vasoactive mediators (5-HT [p=0.017], TXB2 [p=0.0066]); while the treatment group showed a broader regulatory effect, including decreases in LPS (p=0.0124), IL-10 (p=0.0001), and all MMPs (MMP-2 [p=0.0301]). Notably, the treatment intervention had a particularly significant regulatory effect on IL-10 (p=0.0446 compared to the prevention group) and lactate (p=0.0467), but no statistically significant differences were observed in histamine (p=0.3901) and some MMPs.

[0093] Table 2. Serum concentrations of inflammatory markers, matrix metalloproteinases (MMPs), and metabolites in different groups

[0094]

[0095] 4. The effects of amino acid peptide intervention on gut microbiota composition;

[0096] A total of 1,459,468 high-quality reads were obtained from fecal sample sequencing (average 72,973 ± 3,215 reads / sample). Sparse curve analysis confirmed sufficient sampling depth (Good's coverage > 99.8%). Figure 6 Compared with the control group, the α-diversity of gut microbiota in horses with laminitis was significantly reduced (Shannon index: 3.1±0.4 vs 5.2±0.3; p<0.001), while both preventive and therapeutic amino acid peptide interventions restored microbial richness (4.5±0.3 and 4.7±0.3, respectively). β-diversity analysis showed obvious inter-group clustering patterns, and PERMANOVA confirmed significant segregation among groups (R²=0.38, p=0.002) – especially the most significant difference in the microbiome between the laminitis group and the control group.

[0097] At the phylum level, laminitis induced significant changes in the microbiota structure: the relative abundance of Firmicutes increased (86.24% vs. control group 65.75%), while the abundance of Bacteroidetes decreased (2.16% vs. control group 15.30%). Amino acid peptide treatment significantly alleviated this trend (treatment group: Firmicutes 49.75%; Bacteroidetes 20.79%). Figure 4 (A)

[0098] At the genus level, 568 taxa were identified. Laminitis cases exhibited pathogenic overgrowth of *Streptococcus* (41.18% vs. 9.43% in the control group) and *Lactobacillus* (23.31% vs. 0.54% in the control group), accompanied by depletion of beneficial bacteria such as *Akkermansia* (0.86% vs. 4.80% in the control group) and *Lachnospiraceae_UCG-009* (0.19% vs. 4.32% in the control group). Amino acid peptide administration effectively reversed this trend: the abundance of *Akkermansia* increased 20-fold (to 17.24%) in the treatment group, *Streptococcus* decreased 11.8-fold (to 3.48%), and short-chain fatty acid producers (such as *Lachnospiraceae_AC2044_group*, which increased 26.9-fold) were restored. Figure 4 (B) LEfSe analysis identified *Streptococcus equinus* (LDA=4.2) and *Lactobacillus equicursoris* (LDA=3.8) as specific biomarkers for laminitis, while the bacterial flora enriched for amino acid peptides included *Akkermansia muciniphila* and various *Bacteroides* species (B). Figure 7 , Figure 4 (C)

[0099] Correlation analysis showed a strong positive correlation between pathogens and clinical indicators (Sharpea and claudication score: r = 0.72; Streptococcus and IL-17A: r = 0.68), while amino acid peptide-associated flora were negatively correlated with inflammatory markers (Akkermania and MMP-9: r = -0.53). Figure 5Functional predictive analysis revealed upregulation of pro-inflammatory pathways associated with laminitis (peptidoglycan biosynthesis V: log2FC = 2.1) and downregulation of protective metabolic pathways (NAD biosynthesis: log2FC = -1.8). These perturbations partially returned to normal after amino acid peptide treatment. Figure 8 Core microbiome analysis showed limited overlap at the genus level between the control group and the laminitis group (8.48%), which increased to 15.49% after treatment, indicating partial recovery of the control microbiome consortium. Figure 4 (D).

[0100] (vi) Conclusion:

[0101] This study confirms that amino acid peptides can serve as a novel therapy for laminitis, with core applications including preventative interventions for laminitis in racehorses and sport horses, emergency treatment of clinical cases, and comprehensive management of metabolic hoof diseases accompanied by gut microbiota dysbiosis. The amino acid peptides exert their effects through mechanisms such as restoring gut microbiota dysbiosis, inhibiting matrix metalloproteinase (MMP) activity, and reducing inflammatory mediator levels. They show significant improvement in diarrhea and lameness symptoms with good safety, demonstrating therapeutic potential for dysbiosis-related laminitis. Individual microbiome characteristics should be considered in veterinary clinical applications, and amino acid peptides should be used in combination with supportive therapy when systemic inflammation is present. This finding advances the development of laminitis treatment strategies and provides a framework for the development of microbiome-related veterinary therapies. Further research should focus on optimal dosing regimens, long-term safety, and application value in various types of laminitis cases.

[0102] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. An amino acid peptide, characterized in that, The amino acid sequence of the amino acid peptide is shown in SEQ ID NO.1, namely LKAMDPTPPLWIKTQ.

2. The amino acid peptide according to claim 1, characterized in that, The amino acid peptide can regulate the intestinal microbiota of horses, including at least one of the following effects: increasing the relative abundance of Akkermania, increasing the relative abundance of Bacteroidetes, increasing the relative abundance of Trichophyceae, decreasing the relative abundance of Streptococcus, and decreasing the relative abundance of Firmicutes.

3. The use of the amino acid peptide according to claim 1 in the preparation of a medicament for the prevention or treatment of lavage.

4. The application according to claim 3, characterized in that, The prevention or treatment includes improving at least one of the following clinical parameters: increasing fecal pH, reducing Obel claudication score, or improving diarrhea score.

5. The application according to claim 3, characterized in that, The prevention or treatment includes reducing the level of at least one of the following substances in the serum: endotoxin, 5-HT, IL-17A, IL-10, MMP-1, MMP-2, MMP-9.

6. A pharmaceutical composition for the prevention of lavillary dermatitis, characterized in that, It comprises the amino acid peptide as described in claim 1 and sterile physiological saline.

7. The pharmaceutical composition according to claim 6, characterized in that, The drug was administered via intravenous injection into the jugular vein at the following time points: 48 hours before gavage, 24 hours before gavage, and on the day of gavage, with corresponding injection volumes of 150 mL, 150 mL, and 200 mL, respectively.

8. A pharmaceutical composition for treating lavage, characterized in that, It comprises the amino acid peptide as described in claim 1 and sterile physiological saline.

9. The pharmaceutical composition according to claim 8, characterized in that, The medication was administered via jugular vein injection. The first injection was given when the horse showed symptoms of laminitis, such as fever, diarrhea, or an Obel lameness score >1. The second injection was given 12 hours after the first injection. The volume of each injection was 250 mL.

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