Active substance for repairing cat sensitization type barrier and preparation method and application thereof

Abstract

The invention discloses an active substance for repairing a cat sensitization type barrier and a preparation method and application thereof, and relates to the technical field of animal health care products. The cat feed additive comprises one or more of heartleaf houttuynia herb extract, green tea polyphenol, ginkgo leaf extract, glutamine, beta-glucan, linoleic acid, vitamin B6, vitamin B2 and the like, and the components jointly act on oxidative stress, inflammatory response and metabolic process of intestinal tracts and skin of cats. By measuring indexes such as inflammatory factors, oxidative stress and tight junction protein and combining in-vitro metabonomics and transcriptomics analysis, the pharmaceutical composition has remarkable effects in the aspects of oxidation resistance, inflammation resistance, mucosal barrier repair and the like. In addition, the invention also provides application of the active substance in preparation of food for repairing cat barrier dysfunction. Compared with the prior art, an effective health-care strategy can be provided, and cat barrier dysfunction can be remarkably repaired.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of animal health care products, and particularly relates to an active substance for repairing a cat allergen barrier and a preparation method and application thereof. BACKGROUND

[0002] In recent years, allergic skin diseases and accompanying intestinal health problems of cats have become increasingly prominent, and one of the core pathological links is the damage to the intestinal and skin barrier functions and the imbalance of the bidirectional regulation channel between the two, i.e., the "intestine-skin axis". In the prior art, the means for cat allergic diseases are mainly focused on antihistamines, immunosuppressants (such as cyclosporine) or external drugs to relieve symptoms. In view of the limitations of traditional drug treatment, repairing and maintaining the integrity of the intestinal and skin barriers through scientific formulations of functional ingredients has become an important research direction in the field of pet nutrition and health management.

[0003] At present, there are various health supplements for intestinal health on the market, which mainly repair the intestinal mucosal barrier through active peptides, astragalus polysaccharides, glyco-oligosaccharides and other active ingredients. For example, CN 104207151 A discloses a nutritional agent for promoting the recovery of the intestinal function of dogs and cats, and the core components are wheat active peptides, astragalus polysaccharides, glyco-oligosaccharides, vitamins and other components. Similarly, CN 103549410 A proposes a supplement based on marine animal small molecule peptides, glutamine dipeptides, soybean peptides, malt dextrin, vitamins, prebiotics and other components. In addition, CN 112753877 A develops a dog and cat intestinal nutritional supplement composed of glucose, probiotics, genetically engineered lysozyme, vitamins, protease and lipase and other components. However, these existing technologies have obvious limitations: first, the research on the repair mechanism of cat barrier dysfunction is still insufficient, especially the lack of in-depth analysis of key biological processes such as metabolic reprogramming of cat small intestinal epithelial cells and cat skin cells in pathological state and abnormal intracellular signal pathways; there is no active substance that specifically targets the "intestine-skin axis" for multi-target regulation, especially the lack of a natural source solution that can simultaneously repair the intestinal epithelial and skin keratin layer barriers, inhibit excessive immune response and has safety. Therefore, it is of great clinical significance and market value to develop an active substance that can coordinately regulate the intestine-skin axis, restore barrier function through multiple targets and be suitable for the cat body.

[0004] Due to the limitations of current treatments and the insufficient understanding of pathological mechanisms, this invention conceives a novel health care strategy for repairing barrier dysfunction, the core of which lies in the precise regulation of nutrient metabolites. After in-depth research into the metabolic and transcriptional mechanisms of small intestinal epithelial cells and cat skin cells at the cellular level, we successfully identified several key biological metabolic pathways. The regulatory effects of metabolites within these pathways can provide a more comprehensive solution for repairing the integrity of feline barrier dysfunction. Based on this, we have also developed a novel formulation. Summary of the Invention

[0005] In view of this, the present invention aims to propose an active substance for repairing the feline sensitization barrier, its preparation method, and its applications. The changes of the active ingredient in alleviating inflammatory responses, oxidative stress, and enhancing intercellular protein connections were investigated. Subsequently, using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) and RNA sequencing (RNA-seq), the metabolomics and transcriptomics changes in feline small intestinal epithelial cells and feline skin cells were measured. Metabolomics data showed that the active substance significantly affected folic acid biosynthesis and purine metabolism, among other things. Differential metabolite screening identified changes in the content of key metabolites in feline small intestinal epithelial cells and feline skin cells, leading to significant abnormalities in several key metabolic pathways, including purine metabolism, pyrimidine metabolism, taurine and alpha-taurine metabolism, riboflavin metabolism, cofactor biosynthesis, and the pentose phosphate pathway, affecting cells.

[0006] Based on the above findings, the present invention provides an active substance composition for repairing the sensitization barrier in cats, comprising the following components by weight fraction:

[0007]

[0008] The preferred active ingredient composition for repairing the sensitized barrier in cats comprises the following components in weight fractions:

[0009]

[0010] The above-mentioned active substance composition for repairing cat sensitized barriers is used in repairing cat barrier dysfunction.

[0011] The present invention also provides a method for preparing capsules containing the above-mentioned active substances for repairing the feline sensitization barrier, comprising the following steps:

[0012] (1) Weighing and preparing materials: Accurately weigh out the following ingredients: houttuynia cordata extract, green tea polyphenols, ginkgo biloba extract, glutamine, β-glucan, linoleic acid, vitamin B2, vitamin B6, chicken powder, soybean powder, microcrystalline cellulose, silicon dioxide, and magnesium stearate.

[0013] (2) Stirring and sieving: Stir and mix the houttuynia cordata extract, green tea polyphenols, ginkgo biloba extract, glutamine, β-glucan, linoleic acid, chicken powder and soybean powder, and sieve the material through an 80-mesh sieve.

[0014] (3) Mixing: Add 1 / 3 of the microcrystalline cellulose to the stirred and sieved mixture and mix for 10 minutes. Add the remaining 2 / 3 of the filler and silica and continue mixing for 30 minutes. Add magnesium stearate and continue mixing for 5 minutes to ensure thorough mixing.

[0015] (4) Granulation: The mixed material is sent to a semi-automatic capsule filling machine, using enteric-coated empty capsules #3, with a locking pressure of 0.6 Bar;

[0016] (5) Packaging: Pack the soft capsules into finished products and seal them.

[0017] Based on the metabolic and transcriptional mechanisms of feline small intestinal cells and feline skin cells, the effects of each ingredient in this invention are as follows:

[0018] Houttuynia cordata extract, green tea polyphenols, and ginkgo biloba extract can enhance cell vitality, reduce the production of inflammatory factors, enhance antioxidant capacity, and promote the expression of tight junction proteins.

[0019] Glutamine: As the main energy source for intestinal epithelial cells, it supports cell proliferation and maintains the integrity of the mucosal barrier structure.

[0020] β-glucan: activates intestinal immune cells, enhances the secretion of secretory immunoglobulin A (sIgA), and improves the intestinal immune barrier function.

[0021] Linoleic acid: It has a strong anti-inflammatory effect, can regulate the immune response, and helps maintain the normal function of the skin's lipid barrier.

[0022] Vitamin B2 and Vitamin B6: They participate in energy metabolism and the synthesis of antioxidant enzymes, and help maintain the normal metabolic function of epithelial cells.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] 1. Unlike existing technologies used in cosmetics and pharmaceuticals, this invention focuses on the animal food industry. This invention investigates cats at the cellular level, delving into the metabolic and transcriptional changes in feline small intestinal epithelial cells and skin cells. It identifies mechanisms of action related to purine metabolism, pyrimidine metabolism, taurine and linoleic acid metabolism, riboflavin metabolism, cofactor biosynthesis, and the pentose phosphate pathway. By introducing key components such as glutamine, β-glucan, and linoleic acid, this invention provides comprehensive metabolic support for reducing inflammatory cytokine production, enhancing oxidative capacity, and promoting tight junction protein expression in feline small intestinal epithelial cells and skin cells, thereby repairing feline barrier dysfunction.

[0025] 2. The active substances of this invention supplement multiple nutritional metabolism regulators through the intestinal axis, effectively regulating key biochemical pathways in feline small intestinal epithelial cells and feline skin cells. This comprehensive nutritional support helps to fundamentally improve the antioxidant capacity of feline small intestinal epithelial cells and feline skin cells and promote the expression of tight junction proteins, surpassing the limitations of traditional supplements. Attached Figure Description

[0026] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0027] Cat small intestinal epithelial cells (FI) were grouped as follows: blank control group (BC); 2 mg / mL ovalbumin (OVA) treatment group; Houttuynia cordata extract group (HH): 40 μg / mL HH + 2 mg / mL OVA; green tea polyphenol group (GTP): 1 μg / mL GTP + 2 mg / mL; Ginkgo biloba extract group (GBE): 5 μg / mL GBE + 2 mg / mL OVA.

[0028] Feline skin cells (FS) were grouped as follows: BC group was the supernatant of BC group in FI; OVA group was the supernatant of OVA in FI; HH group was the supernatant of HH in FI; GTP group was the supernatant of GTP in FI; and GBE group was the supernatant of GBE in FI.

[0029] Figure 1 Effects of HH, GTP, and GBE interventions on the levels of inflammatory factors in FI and FS. (A) Interleukin-1β (IL-1β) level in FI; (B) Interleukin-6 (IL-6) level in FI; (C) Tumor necrosis factor-α (TNF-α) level in FI; (D) IL-1β level in FS; (E) IL-6 level in FS; (F) TNF-α level in FS.

[0030] Figure 2 Effects of HH, GTP, and GBE interventions on oxidative stress indices in FI and FS. (A) Superoxide dismutase (SOD) activity in FI; (B) Propylene glycol (MDA) content in FI; (C) Reduced glutathione (GSH) content in FI; (D) Catalase (CAT) activity in FI; (E) SOD activity in FS; (F) MDA content in FS; (G) GSH content in FS; (H) CAT activity in FS.

[0031] Figure 3Effects of HH, GTP, and GBE interventions on mucin content in FI and FS. (A) Mucin (MUC-1) content in FI; (B) Mucin (MUC-2) content in FI; (C) Mucin (MUC-3) content in FI; (D) MUC-1 content in FS; (E) MUC-2 content in FS; (F) MUC-3 content in FS.

[0032] Figure 4 Effects of HH, GTP, and GBE interventions on the content of tight junction proteins in FI and FS. (A) Content of tight junction protein (Occludin) in FI; (B) Content of tight junction protein 1 (ZO-1) in FI; (C) Content of blocking protein (Claudin) in FI; (D) Content of Occludin in FS; (E) Content of ZO-1 in FS; (F) Content of Claudin in FS.

[0033] Figure 5 Effects of HH, GTP, and GBE interventions on skin protein content in FS. (A) Filamentin (FLG) content in FS; (B) Lobelin (LOR) content in FS.

[0034] Figure 6 Volcano plots and cluster heatmaps of differentially expressed metabolites among groups in FI and FS. (AB) Volcano plots of differentially expressed metabolites among groups in FI; (C) Cluster heatmaps of differentially expressed metabolites among groups in FI; (DE) Volcano plots of differentially expressed metabolites among groups in FS; (F) Cluster heatmaps of differentially expressed metabolites among groups in FS.

[0035] Figure 7 Volcano plots and cluster heatmaps of differentially expressed genes among groups in FI and FS. (AB) Volcano plots of differentially expressed genes among groups in FI; (C) Cluster heatmaps of differentially expressed genes among groups in FI; (DE) Volcano plots of differentially expressed genes among groups in FI; (F) Cluster heatmaps of differentially expressed genes among groups in FI. Detailed Implementation

[0036] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0037] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0038] The extracts of Houttuynia cordata (containing 1% to 3% flavonoids by mass) and green tea polyphenols (containing 90% to 98% tea polyphenols by mass) of this invention were purchased from Shaanxi Benhe Biotechnology Co., Ltd., and the extract of Ginkgo biloba (containing 20% ​​to 28% flavonoids by mass) was purchased from Xi'an Mumeng Biological Products Co., Ltd.

[0039] Example 1

[0040] Cell culture

[0041] FI or FS were cultured in M-3 medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics at 37°C and 5% CO2 until 80% confluence. The cells were then digested with trypsin and the cell concentration was adjusted to 1×10⁻⁶. 5 Cells were seeded at 2 mL / well in 6-well plates and cultured for 24 h. Cells were then washed twice with phosphate-buffered saline (PBS, pH 7.4) and cultured for another 24 h as follows: FI: blank control group (serum-free DMEM, BC group), negative control group (2 mg / mL ovalbumin, OVA), and three experimental groups (2 mg / mL OVA combined with 40 μg / mL HH, 1 μg / mL GTP, or 5 μg / mL GBE, respectively); FS: cells were treated with the supernatant from the corresponding groups in FI, i.e., BC group, OVA group, HH group, GTP group, and GBE group. After 24 h of culture, the supernatant from all groups was collected, and cells were lysed using Western blotting and IP cell lysis buffer for subsequent analysis.

[0042] Example 2

[0043] Inflammatory factor marker measurement

[0044] Cells were treated according to the method in Example 1, and the supernatant from FI and FS was collected. Specifically, the operation was performed according to the instructions of the IL-1β, IL-6 and TNF-α enzyme-linked immunosorbent assay (ELISA) kit, and the absorbance value at a wavelength of 450 nm was measured using an ELISA reader.

[0045] Experimental results showed that HH, GTP, and GBE could effectively alleviate the inflammatory response induced by OVA in fibrillation (FI) and fibrosis (FS). Figure 1As shown, compared with the BC group, OVA stimulation significantly increased the expression levels of inflammatory cytokines such as IL-1β, IL-6, and TNF-α in FI and FS (P < 0.05). However, after intervention with HH, GTP, and GBE, the levels of these inflammatory cytokines all decreased significantly (P < 0.05), with HH showing the strongest anti-inflammatory effect in FI and GBE showing the strongest anti-inflammatory effect in FS. These findings confirm that HH, GTP, and GBE can exert cytoprotective effects by regulating the expression of inflammatory cytokines and inhibiting OVA-induced cellular inflammatory responses.

[0046] Example 3

[0047] Oxidative stress index measurement

[0048] Cells were treated according to the method in Example 1, and cell lysates were collected after 24 hours of culture. MDA condensed with thiobarbituric acid (TBA) to form a red product, and the specific procedure was performed according to the kit instructions. The absorbance was measured at 532 nm using a microplate reader. GSH reacted with dithiodinitrobenzoic acid (DTNB) to form a yellow compound, and the GSH content was measured at 405 nm. The reaction of CAT to decompose hydrogen peroxide could be rapidly terminated by adding ammonium molybdate. The remaining hydrogen peroxide reacted with ammonium molybdate to produce a pale yellow complex, and the absorbance was measured at 405 nm using a microplate reader. SOD inhibited the formation of formazan by dismutating superoxide anions, and SOD activity could be indirectly measured by measuring changes in absorbance at 450 nm.

[0049] Experimental results showed that HH, GTP, and GBE had significant protective effects against oxidative stress induced by OVA in fibrillation (FI) and fibrosis (FS). Figure 2 As shown, compared with the BC group, OVA treatment significantly reduced GSH content, SOD and CAT activity in FI and FS (P < 0.05), while significantly increasing MDA content (P < 0.05), indicating that OVA successfully induced cellular oxidative stress damage. Notably, after intervention with HH, GTP, or GBE, GSH content, SOD and CAT activity in cells significantly recovered (P < 0.05), while MDA level significantly decreased (P < 0.05). These results fully demonstrate that HH, GTP, and GBE can effectively alleviate OVA-induced oxidative stress damage in FI and FS by enhancing the antioxidant defense system and reducing lipid peroxidation.

[0050] Example 4

[0051] Mucin assay

[0052] Cell lysates (FI) and fibroblasts (FS) were treated according to the experimental method described in Example 1. After 24 hours of culture, cell lysates were collected for mucin detection. Mucins (MUCs), as a class of high-molecular-weight glycoproteins, are key components in maintaining the structure and function of the intestinal mucosal barrier. This experiment used ELISA to determine the expression levels of MUC-1, MUC-2, and MUC-3, strictly following the operating procedures of the ELISA kits specific to each mucin subtype. Specifically, after reacting the cell lysates with the antibodies provided in the kit, the absorbance was measured at 450 nm using a microplate reader to quantitatively analyze the differences in expression of different mucins in each group of cells.

[0053] Experimental results show that HH, GTP, and GBE can effectively protect the mucosal barrier function induced by OVA in FI and FS. Figure 3 As shown, compared with the BC group, OVA treatment significantly reduced the expression levels of MUC-1, MUC-2, and MUC-3 (P < 0.05), indicating that OVA disrupted the integrity of the intestinal and skin barriers. However, after intervention with HH, GTP, and GBE, the expression of all three mucins was significantly restored (P < 0.05). These data confirm that HH, GTP, and GBE can enhance cell barrier function by upregulating mucin expression, thereby effectively alleviating OVA-induced damage to the intestinal and skin barriers. This protective effect may be related to its anti-inflammatory and antioxidant mechanisms, providing experimental evidence for the application of natural products in maintaining intestinal health.

[0054] Example 5

[0055] Tight junction protein assay

[0056] Cell lysis (FI) and fibroblast lysis (FS) were treated according to the experimental method in Example 1. After 24 hours of culture, cell lysates were collected for tight junction protein detection. Tight junction proteins are core components of epithelial cell tight junctions and are crucial for maintaining the integrity and selective permeability of the mucosal barrier. Occludin, ZO-1, and Claudin are the most representative proteins. This experiment used ELISA to determine the expression levels of Occludin, ZO-1, and Claudin, strictly following the operating procedures of the ELISA kit. Specifically, after reacting the cell lysates with the antibody provided in the kit, the absorbance was measured at 450 nm using a microplate reader to quantitatively analyze the differences in tight junction protein expression among the groups of cells.

[0057] Experimental data clearly show that HH, GTP, and GBE have significant protective effects against the tight junction barrier induced by OVA in FI and FS. Figure 4As shown, compared with the BC group, OVA stimulation significantly downregulated the expression levels of key tight junction proteins Occludin, ZO-1, and Claudin (P < 0.05), directly confirming that OVA treatment disrupts the tight junction structure of intestinal epithelial cells and skin cells. Notably, after treatment with HH, GTP, and GBE, the expression levels of these three core proteins maintaining the cellular bypass barrier all significantly rebounded (P < 0.05). These findings not only confirm the protective effects of the three natural active ingredients on the intestinal and skin physical barriers at the molecular level, but also reveal their mechanism of action in repairing OVA-induced intestinal and skin barrier damage by specifically regulating the expression of tight junction proteins, providing important theoretical basis for the application of natural products in maintaining intestinal and skin health.

[0058] Example 6

[0059] Skin protein assay

[0060] FI and FS were treated according to the experimental method in Example 1, and cell lysates were collected after 24 hours of culture for skin protein detection. Filamentin and lipohypophysin are key proteins produced by epidermal cells, crucial for maintaining the integrity and selective permeability of the skin barrier. This experiment used ELISA to determine the expression levels of FLG and LOR, strictly following the operating procedures of the ELISA kit. Specifically, after reacting the cell lysates with the antibody provided in the kit, the absorbance was measured at 450 nm using a microplate reader to quantitatively analyze the differences in tight junction protein expression among the groups of cells.

[0061] Experimental data showed that HH, GTP, and GBE had significant protective effects on skin barrier function induced by OVA in FI and FS cells. Figure 5 As shown, compared with the BC group, OVA stimulation significantly downregulated the expression levels of skin barrier proteins FLG and LOR (P<0.05), indicating that OVA treatment disrupts the intercellular connections in skin cells. Notably, after intervention with HH, GTP, or GBE, the expression of FLG and LOR significantly rebounded (P<0.05). These results suggest at the molecular level that these three natural active ingredients can effectively alleviate OVA-induced skin barrier damage, and their mechanism of action is related to the specific regulation of the expression of key skin barrier proteins. This finding provides important theoretical basis for the application of natural products in skin health maintenance.

[0062] Example 7

[0063] To gain a deeper understanding of the effects of the three plant extracts on FI and FS, HH was used as an example for FI, and GBE was used as an example for FS. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used to perform detailed metabolomics analysis of FI and FS. Cells were treated according to the method in Example 1 and cultured for 24 h.

[0064] In the FI model, metabolomics analysis revealed the regulatory role of HH on OVA-induced FI metabolic disorders. For example... Figure 6 As shown, the volcano plot and cluster heatmap results indicate that, compared with the BC group, the OVA treatment group showed significant metabolic reprogramming, with 206 metabolites upregulated and 55 metabolites downregulated; while after HH intervention, 12 metabolites were upregulated and 26 were downregulated. These differentially expressed metabolites mainly involve the following key pathways: (1) pyrimidine metabolism (thymine, 2'-deoxyuridine; thymidine, 5'-monophosphateuridine, L-glutamine); (2) taurine and taurine metabolism (taurine, taurocholic acid); (3) riboflavin metabolism (flavin mononucleotide, photopigment); (4) nitrogen metabolism (L-glutamine, L-glutamate). Further enrichment analysis using the Kyoto Encyclopedia of Genetics and Genomes (KEGG) revealed that the regulatory role of HH is mainly achieved through pathways such as folic acid biosynthesis and purine metabolism, with mechanisms involving the regulation of key metabolite levels (such as 7,8-dihydroneopterin, inosine-5'-bisphosphate, and uric acid). These findings elucidate at the metabolic level that HH effectively corrects OVA-induced metabolic imbalances through multi-target and multi-pathway synergistic effects, thereby alleviating cell damage and promoting barrier function repair.

[0065] In the FS model, metabolomics analysis showed that GBE had a significant regulatory effect on OVA-induced metabolic disorders. Figure 6As shown in the volcano plot and cluster heatmap results, compared with the BC group, the OVA group showed significant metabolic reprogramming, with a total of 467 metabolites upregulated and 303 metabolites downregulated; while after GBE intervention, the metabolic changes tended to be milder, with only 19 metabolites upregulated and 57 metabolites downregulated. These differential metabolites are mainly enriched in the following key pathways: (1) cofactor biosynthesis (involving S-adenosine-L-methionine, vitamin B5, L-glutamic acid, etc.); (2) lysine biosynthesis (including L-aspartic acid, L-lysine, L-2-aminoadipic acid, etc.); (3) histidine metabolism (covering L-glutamic acid, 5-aminoimidazolium-4-carboxamide-1-β-D-furanoside-5'-monophosphate, N-carbamoylglutamic acid, etc.); (4) pentose phosphate pathway (including D-gluconic acid, D-erythrose-4-phosphate, fructose phosphate, deoxyribose-1-phosphate); (5) β-alanine metabolism (involving β-alanyl-L-lysine, L-aspartic acid, β-alanyl-L-arginine, etc.). KEGG enrichment analysis further revealed that the regulatory effect of GBE is mainly achieved through pathways such as nicotinic acid and nicotinamide metabolism, glutathione metabolism, and glycerophospholipid metabolism. Its mechanism is closely related to the restoration of levels of key metabolites (such as nicotinic acid mononucleotide and γ-glutamyl alanine). These results elucidate from a metabolic perspective that GBE can effectively reverse the metabolic imbalance induced by OVA through multi-target and multi-pathway synergistic regulation, thereby alleviating cell damage and promoting barrier function repair.

[0066] Example 8

[0067] To gain a deeper understanding of the effects of the three plant extracts on FI and FS, HH was used as an example in FI, and GBE was used as an example in FS. Transcriptomic sequencing of cellular RNA was performed using the Illumina sequencing platform. Cells were treated according to the method in Example 1 and cultured for 24 hours. An equal volume of sample was taken from each experimental sample and mixed to serve as a quality control group. RNA was extracted from each group using standard methods, libraries were constructed, and sequencing was performed. Differential expression analysis between the two comparison combinations was conducted using DESeq2 software (1.20.0).

[0068] In the FI model, the gene expression analysis results are as follows: Figure 7As shown in the figure, compared with the BC group, the OVA treatment group showed 269 upregulated genes and 245 downregulated genes; while compared with the OVA group, the HH treatment group showed 24 upregulated genes and 1252 downregulated genes, indicating that HH can significantly reverse the transcriptome changes induced by OVA. Through gene ontology (GO) and KEGG enrichment analysis of the OVA-induced FI damage model, we systematically revealed its molecular mechanism and the intervention effect of HH. GO analysis showed that OVA treatment was significantly enriched in three major functional categories: in biological processes (BP), mainly involving immune system processes and immune responses; in cellular components (CC), concentrated in structures such as nucleosomes, protein-DNA complexes, chromatin, and chromosomes; and in molecular functions (MF), enriched in items related to signal receptor binding, receptor regulatory activity, chemokines, and cytokine activity. Following HH intervention, the aforementioned functional categories underwent significant alterations, particularly in post-translational protein modifications, nuclear and chromosome-related structures, and the binding and catalytic activity of ubiquitination systems and transcription factors, exhibiting characteristic regulation. KEGG pathway analysis further corroborated these findings: the OVA-treated group showed significant enrichment in disease- and immune-related pathways (such as the IL-17 and TNF signaling pathways), indicating that OVA disrupts the integrity of the intestinal epithelial barrier by activating inflammatory responses and releasing cytotoxicity. Conversely, the HH intervention group was primarily enriched in DNA repair and cellular transport pathways (such as homologous recombination and nucleoplasmic transport), suggesting that HH may effectively alleviate OVA-induced intestinal epithelial damage by enhancing DNA repair capabilities and intracellular transport mechanisms. In summary, this study reveals the therapeutic potential of HH in repairing the intestinal epithelial barrier from a systems biology perspective, providing a theoretical basis for developing natural product-based intestinal protection strategies.

[0069] In the FS cell model, the gene expression profiling results are as follows: Figure 7As shown in the figure, compared with the BC group, the OVA treatment group showed significant upregulation of 3,637 genes and significant downregulation of 3,607 genes; while compared with the OVA model group, the GBE intervention group showed upregulation of 363 genes and downregulation of 326 genes. Through GO and KEGG pathway enrichment analysis of the OVA-induced FS cell damage model, we systematically revealed its potential molecular mechanisms and the interventional effect of GBE. GO enrichment analysis showed that OVA treatment mainly affected three functional categories: at the BP level, it significantly enriched in pathways related to protein phosphorylation, cell surface receptor signaling, and cell differentiation; at the CC level, it mainly concentrated on items related to nuclear structure; and at the MF level, it involved kinase and phosphorylation activity, methyltransferase activity, and hydrolase activity. Notably, these functional categories were significantly altered after GBE treatment, especially showing characteristic enrichment in biosynthetic processes, methylation modification, microtubule cytoskeleton structure, and dynein complex, suggesting that GBE may exert a protective effect by regulating these key biological processes. The KEGG pathway analysis results were highly consistent with the GO analysis. Compared to the BC group, the OVA-treated group showed significant enrichment in multiple cancer pathways, viral infection pathways, TNF signaling pathways, and cellular senescence pathways, indicating that OVA may induce skin cell damage by activating abnormal immune responses and chronic inflammatory reactions, thereby disrupting cell connections and barrier integrity. In contrast, the GBE intervention group showed significant enrichment in key pathways such as the PI3K-AKT signaling pathway, FOXO signaling pathway, and TNF signaling pathway, suggesting that GBE may effectively reverse OVA-induced skin barrier damage through multi-target and multi-pathway synergistic regulation, enhancing intercellular connections, inhibiting inflammatory responses, and promoting cellular homeostasis restoration. In conclusion, this study elucidates the potential mechanism of GBE in alleviating OVA-induced skin damage from a systems biology perspective, providing a solid theoretical basis for its application as a natural product in the development of skin barrier protectants.

[0070] Examples 9-11

[0071] Examples 9-11: Capsules were prepared according to the formulations shown in Table 1 and in the following manner:

[0072] (1) Weighing and preparing materials: Accurately weigh out the following ingredients: houttuynia cordata extract, green tea polyphenols, ginkgo biloba extract, glutamine, β-glucan, linoleic acid, vitamin B2, vitamin B6, chicken powder, soybean powder, microcrystalline cellulose, silicon dioxide, and magnesium stearate.

[0073] (2) Stirring and sieving: Stir and mix the houttuynia cordata extract, green tea polyphenols, ginkgo biloba extract, glutamine, β-glucan, linoleic acid, chicken powder and soybean powder, and sieve the material through an 80-mesh sieve.

[0074] (3) Mixing: Add 1 / 3 of the microcrystalline cellulose to the stirred and sieved mixture and mix for 10 minutes. Add the remaining 2 / 3 of the filler and silica and continue mixing for 30 minutes. Add magnesium stearate and continue mixing for 5 minutes to ensure thorough mixing.

[0075] (4) Granulation: The mixed material is sent to a semi-automatic capsule filling machine, using enteric-coated empty capsules #3, with a locking pressure of 0.6 Bar;

[0076] (5) Packaging: Pack the soft capsules into finished products and seal them.

[0077] Comparative Examples 1-2

[0078] Comparative Examples 1-2 were prepared according to the formulations shown in Table 1. The preparation process, including weighing and preparing materials, granulation and drying, and total mixing and packaging, was the same as in Examples 9-11 above. The values ​​in Table 1 are in grams. Each capsule weighed approximately 300 ± 10 mg, and a total of 200 soft capsules were prepared.

[0079] Table 1. Formulations used in each embodiment and comparative example (unit: g)

[0080]

[0081] Test case

[0082] 1. Test Methods

[0083] (1) Experimental animals and grouping: 40 cats with diarrhea symptoms were selected and randomly divided into 5 groups of 8 cats each.

[0084] 2. Intervention Program: Groups 1-3 were the experimental groups, fed a basal diet daily and simultaneously given capsules prepared according to treatment 9-11 daily. Groups 4-5 were the control groups, fed a basal diet daily and simultaneously given capsules prepared according to control 1-2 daily. The dosage for each group was one capsule once daily for 15 days. Activity levels were observed.

[0085] 3. Test Results

[0086] The effects on each group of cats are shown in Table 2. Compared with the control group, feeding cats with Examples 1-3 improved their mental and skin condition and effectively reduced the incidence of diarrhea.

[0087] Table 2 Results of barrier dysfunction index measurement in each group of cats

[0088]

[0089] Note: Mental state score: ▲: Lethargic (sleepiness, loss of appetite); ▲▲: Fair (reduced activity); ▲▲▲: Good (normal activity); ▲▲▲▲: Active (energetic); ▲▲▲▲▲: Very active (daily assessment for each cat, average of the group values).

[0090] Skin condition rating: ●: Very severe (pyoderma, hyperpigmentation, severe ulceration); ●●: Severe (extensive hair loss, oozing and erosion, severe crusting); ●●●: Moderate (obvious erythema, hair loss, papules); ●●●●: Mild (mild erythema, mild hair loss); ●●●●●: Normal (dense, glossy coat) (Assess each cat daily, average group value).

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An active substance composition for repairing the allergenic barrier of cats, characterized in that consists of the following weight fractions of components:

2. An active substance composition for repairing cat allergen barrier according to claim 1, characterized in that consists of the following weight fractions of components:

3. Process for the preparation of a capsule containing an active substance composition with a repair cat allergen barrier according to claim 1 or 2, characterized in that The following steps are carried out: (1) weighing raw materials: accurately weigh the extract of Houttuynia cordata, green tea polyphenol, Ginkgo biloba extract, glutamine, beta-glucan, linoleic acid, vitamin B2, vitamin B6, chicken powder, soybean powder, microcrystalline cellulose, silicon dioxide, magnesium stearate for standby; (2) stirring and sieving: stir and mix the extract of Houttuynia cordata, green tea polyphenol, Ginkgo biloba extract, glutamine, beta-glucan, linoleic acid, chicken powder and soybean powder, and pass the materials through an 80-mesh sieve; (3) mixing: add 1 / 3 of the microcrystalline cellulose to the stirred and sieved mixture and mix for 10 minutes, add the remaining 2 / 3 of the filler and silicon dioxide and continue mixing for 30 minutes, add magnesium stearate and continue mixing for 5 minutes, and mix thoroughly; (4) granulation: send the mixed material to a semi-automatic capsule filling machine, use enteric hollow capsules #3, and the locking pressure is 0.6 Bar; (5) packaging: package the soft capsules into finished products and seal them.

4. The active substance composition for repairing the sensitized barrier of cats according to any one of claims 1-2, for use in repairing the barrier dysfunction of cats.

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