Evaluation method of bile acids in alleviating intestinal inflammation in spotted acorns

Abstract

This invention discloses an evaluation method for bile acids in alleviating intestinal inflammation in grouper, belonging to the field of aquaculture technology. Addressing the intestinal inflammation problem caused by feed oxidation, pathogen infection, or water quality deterioration in intensive grouper farming, this invention proposes to achieve efficient regulation of intestinal health by adding a compound bile acid composition to the feed. The core technical solution of the method is as follows: a compound bile acid is formed by compounding porcine deoxycholic acid, chenodeoxycholic acid, and porcine cholic acid in a specific ratio, combined with a carrier premixing process to prepare a bile acid premix, which is then added to the basal feed at 0.15%-0.60% of the total feed mass. This invention effectively alleviates enteritis symptoms in grouper by enhancing the integrity of the intestinal epithelial structure, repairing damaged mucosal barriers, regulating inflammatory responses, and optimizing intestinal flora composition. It provides a safe and efficient technical solution for replacing feed antibiotics in aquaculture, with significant economic and ecological benefits.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, specifically to an evaluation method for bile acids in alleviating intestinal inflammation in the starling. Background Technology

[0002] Compared to mammals, fish have a relatively underdeveloped intestinal immune system, making them more susceptible to disease in intensive aquaculture. Enteritis, in particular, has become one of the most serious diseases in farmed fish, often leading to high mortality rates and significant economic losses. The occurrence of fish enteritis is the result of multiple interacting factors, including excessively high stocking densities, pathogen invasion, deterioration of the aquaculture environment, and lack of nutritional components in feed. The development of enteritis involves a complex pathological process involving multiple factors. Existing research indicates that its pathogenesis is closely related to key aspects such as intestinal barrier dysfunction, immune homeostasis imbalance, oxidative stress, and gut microbiota dysbiosis. In aquaculture, enteritis not only reduces feeding efficiency and growth performance in fish but also impairs immune defense, potentially inducing large-scale mortality and causing enormous economic losses to the aquaculture industry.

[0003] The Chinese red grouper, an important economic fish, plays a vital role in Hainan's aquaculture industry. However, with the expansion of farming scale and the increase in intensification, fish diseases, especially digestive system diseases such as intestinal inflammation, are becoming increasingly serious, severely impacting farming efficiency and fish health. Intestinal inflammation not only leads to reduced feed intake and slow growth but can also trigger secondary infections and even cause large-scale mortality. Currently, the prevention and treatment of enteritis in Chinese red grouper mainly rely on chemical drugs and antibiotics. However, long-term excessive use of these drugs may damage the fish's intestinal mucosal immune system, further reducing its disease resistance. Therefore, finding natural, safe, and highly effective additives to alleviate fish enteritis as alternatives to feed antibiotics has become an important research direction in the aquaculture field. Summary of the Invention

[0004] The purpose of this invention is to provide an evaluation method for bile acids in alleviating intestinal inflammation in starlings. This method can effectively reduce intestinal inflammation in starlings, improve their intestinal health and growth performance, and is safe with no side effects, thereby improving aquaculture efficiency.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for evaluating the role of bile acids in alleviating intestinal inflammation in *Pleurotus ostreatus*, comprising the following steps: S1. Inducing intestinal inflammation: A basic feed was formulated using fish meal, chicken meal, soy protein concentrate, flour, squid paste, fish oil, soybean oil, multivitamins, minerals and cellulose as raw materials. Chemical inducers were added to the basic feed to induce typical enteritis symptoms and construct an intestinal inflammation model of juvenile grouper. S2. Formulating bile acid feed: Add different levels of bile acids to the basic feed to formulate bile acid feed; S3. Feeding method: Juvenile East Star Grouper with enteritis symptoms were used as the experimental group and fed with feed containing bile acids. Healthy juvenile East Star Grouper were used as the control group and fed with basic feed without added bile acids. The feeding period was four weeks, and the fish were fed twice a day until they were full. S4. After feeding, all experimental fish were fasted for 24 hours. The number of fish in each net cage was counted and weighed. The weight gain rate was calculated. Six fish were randomly selected from each net cage and anesthetized with 50 mg / L tricaine mesylate. Their body weight, intestinal weight and length were measured. The relative intestinal weight, relative intestinal length and Zihler index were calculated. S5. Sampling Analysis: Histopathological observation of intestinal tissue: Midgut samples were taken from 3 fish, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned and stained with hematoxylin and eosin to observe intestinal morphological indicators. Inflammation-related enzyme activity detection: Midgut samples from 3 fish were collected, stored in liquid nitrogen, and tissue homogenates were prepared to detect myeloperoxidase and β-N-acetylglucosidase activities. Analysis of inflammation-related gene expression: Total RNA was extracted from midgut samples preserved in liquid nitrogen, reverse transcribed into cDNA, and the mRNA expression levels of pro-inflammatory and anti-inflammatory factors were measured by real-time quantitative PCR. Gut microbial diversity analysis: Hippu samples were taken from 3 fish, flash-frozen in liquid nitrogen, and microbial genomic DNA was extracted. The structure and diversity of the microbial community were analyzed by high-throughput sequencing of the V3-V4 region of the 16S rRNA gene.

[0006] Furthermore, the chemical inducing agent is 0.75% sodium dextran sulfate.

[0007] Furthermore, the different levels of bile acids include addition amounts of 0.00%, 0.15%, 0.30%, 0.45%, and 0.60% of the basic feed weight.

[0008] Secondly, the present invention provides the application of bile acids in the preparation of products that alleviate intestinal inflammation of spotted lentigines.

[0009] Furthermore, the amount of bile acids added is 0.15%-0.60% of the basal feed mass, and the total mass of bile acids contains ≥68% porcine deoxycholic acid, ≥17% chenodeoxycholic acid and ≥9% porcine cholic acid.

[0010] Furthermore, the bile acids alleviate intestinal inflammation through the following pathways: Upregulates the expression of tight junction protein genes (ZO-1, Occludin) and enhances intestinal barrier function; It inhibits the mRNA expression of pro-inflammatory factors TNF-α and IL-1β, and promotes the expression of anti-inflammatory factor IL-10; It regulates the structure of the gut microbiota, enriches Firmicutes and inhibits Proteobacteria to optimize the gut microbiota structure.

[0011] Furthermore, the bile acids can improve intestinal morphology parameters of the spotted dinoflagellate, including increasing the height of small intestinal villi, the thickness of the muscular layer, and the number of goblet cells.

[0012] Furthermore, the bile acids reduce the activity of intestinal inflammation-related enzymes, including myeloperoxidase and β-N-acetylglucosidase.

[0013] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects: This invention intervened in DSS-induced enteritis in *Stachys pubescens* by adding different levels of bile acids (0.15%-0.60%). The results showed that adding 0.60% bile acids had the best effect on improving gut health. This reveals the potential role of bile acids in alleviating DSS-induced enteritis and improving gut health in *Stachys pubescens*. The results indicated that bile acids significantly improved intestinal morphology, increasing villus height, enhancing muscular layer thickness, and improving overall structural integrity. These improvements were closely related to the upregulation of tight junction proteins (e.g., ZO-1 and Claudin-1), which play a crucial role in maintaining intestinal barrier function and regulating permeability. Furthermore, bile acids also alleviated intestinal inflammation by downregulating the expression of pro-inflammatory cytokines (e.g., TNF-α and IL-1β) and upregulating the expression of anti-inflammatory cytokines (e.g., IL-10). Simultaneously, bile acids also regulated the composition of the gut microbiota, increasing the proportion of beneficial Firmicutes and inhibiting the excessive proliferation of harmful Proteobacteria. Therefore, as a feed additive, bile acids have the potential to promote gut health in aquaculture and can become an effective strategy for improving fish health by reducing intestinal lesions and improving growth performance. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0015] Figure 1 The graph shows the effect of bile acids on the growth performance of DSS-induced enteritis-inducing stellate ternata. A: final body weight; B: weight gain rate. Figure 2 The graph shows the effect of bile acids on the intestinal index of DSS-induced enteritis in the stellate foetida. A: relative intestinal length; B: relative intestinal weight; C: Zihler's index. Figure 3 Histopathological observation of the midgut of the star scorpion; Figure 4 The diagram shows the effect of bile acids on the intestinal morphology of DSS-induced enteritis in patients with stellate follicle syndrome. A: VH (intestinal villi height); B: VW (intestinal villi width); C: MT (muscular layer thickness); D: GC (goblet cell number). Figure 5 The figure shows the effect of dietary bile acids on the activities of intestinal myeloperoxidase (MPO) and β-N-acetyl-D-glucosidase (NAG) in DSS-induced enteritis stellate lentigines. A: Myeloperoxidase (MPO) activity; B: β-N-acetyl-D-glucosidase (NAG) activity. Figure 6 Figure showing the effect of bile acids on the expression of intestinal inflammatory cytokine genes in DSS-induced enteritis in *Stellaria media*. Figure 7 Figure showing the effect of bile acids on the expression of tight junction protein genes in the gut of DSS-induced enteritis in *Stellaria media*. Figure 8 Analysis of intestinal microbial diversity in *D. stellaria media* induced by bile acids; A: Venn diagram; B: alpha diversity analysis. Detailed Implementation

[0016] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0017] The objective of this invention is achieved through the following technical solution: I. Experimental Design The diet used in this experiment was based on a basal diet (as shown in Table 1), supplemented with 0.75% sodium dextran sulfate (DSS) and different levels of bile acids (0.00%, 0.15%, 0.30%, 0.45%, and 0.60%). Sodium dextran sulfate was purchased from Shanghai Yuanye Biotechnology Co., Ltd., and the bile acids (68% porcine deoxycholic acid, 17% chenodeoxycholic acid, and 9% porcine cholic acid) were purchased from Shandong Longchang Animal Health Products Co., Ltd.

[0018] Juvenile grouper were purchased from Linlan Aquaculture Co., Ltd., Lantian Village, Dong'ao Town, Wanning City, Hainan Province. After one week of acclimatization to commercial feed, 360 juveniles of similar size (initial average weight 15.13±0.10g) were selected and randomly assigned to 18 floating net cages (75cm×50cm×45cm), which were placed in an indoor cement pond (4m×3m×1.5m). An enteritis model was established by feeding the fish with 0.75% DSS feed for one week, followed by three weeks of feeding with diets containing gradient levels of bile acids. The experimental groups were named DSSBA15, DSSBA30, DSSBA45, and DSSBA60; the DSS model group was DSSBA0 (0.75% DSS feed, no bile acids); and the control group (control) did not receive DSS treatment. Each group had three replicate net cages, with 20 fish per cage. Fish were fed twice daily (8:30 and 16:30) using a satiating feeding method. Daily monitoring of water temperature (30±0.5℃), total ammonia nitrogen (<0.10mg / L), and dissolved oxygen (6.4±0.2mg / L).

[0019] Table 1. Formulation and Nutritional Composition Analysis of Basic Feed

[0020] II. Effectiveness Evaluation Growth performance determination: After the experiment, all experimental fish were fasted for 24 hours, and the number and weight of fish in each net cage were counted and calculated to determine the weight gain rate (WGR). Six fish were randomly selected from each net cage and anesthetized with 50 mg / L tricaine mesylate for the Chinese star grouper. Their body weight, intestinal weight, and length were measured, and the relative intestinal weight (RIW), relative intestinal length (RIL), and Zihler index (ZI) were calculated.

[0021] Weight gain rate (WG%) = (Final weight (FBW, g) - Initial weight (IBW, g) / Initial weight (IBW, g) × 100; Relative intestine weight (RIW%) = Intestine weight (g) / Body weight (g); Relative intestine length (RIL%) = Intestine length (cm) / Body length (cm); Zihler's Star Spot Index (ZI) = Intestine length (mm) / Star Spot × (body weight (g) 1 / 3); The Hartley test was used to assess the normality and homogeneity of the data. Subsequently, one-way ANOVA and Duncan's multiple range test (SPSS East Star Spot 22.0, USA) were performed to determine whether there were significant differences between the different treatments.

[0022] Histopathological observation of intestinal tissue: Midgut samples were collected from three fish and fixed with 4% paraformaldehyde. The tissue was removed from the fixative and trimmed using a scalpel in a fume hood. The trimmed midgut tissue and corresponding labels were placed in embedding frames. The dehydration box was placed in a dehydrator and dehydrated sequentially using a gradient of alcohols: 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, 65°C melted paraffin I for 1 hour, 65°C melted paraffin II for 1 hour, and 65°C melted paraffin III for 1 hour. The paraffin-soaked midgut tissue was then embedded in an embedding machine. First, the melted paraffin was placed in the embedding frame. Before the paraffin solidified, the midgut tissue was removed from the dehydration box, placed in the embedding frame according to the embedding surface requirements, and labeled accordingly. The paraffin blocks were cooled to -20°C on a freezing stage. After solidification, they were removed from the embedding frame and trimmed. The trimmed blocks were then sectioned using a paraffin microtome to a thickness of 4 μm. The sections were floated on a 40°C warm water spreader to flatten the tissue, then lifted onto a glass slide and baked in a 60°C oven. The sections were then removed after the paraffin had melted and the water had dried. The sections were subsequently stained with hematoxylin and eosin (H&E) for observation and image acquisition under a microscope equipped with a digital camera (Olympus IX 71, Japan). Morphological parameters such as villus height, villus width, muscle layer thickness, and goblet cell number were measured using ImageJ image analysis software.

[0023] Assay for intestinal inflammation-related enzyme activity: Foregut tissue samples were accurately weighed and homogenized in pre-cooled phosphate-buffered saline (PBS, pH 7.4) at a weight-to-volume ratio of 1:19 (w / v) to prepare a 5% tissue homogenate suspension (without centrifugation). Myeloperoxidase (MPO) activity in the homogenate was measured using the Nanjing Jiancheng Bioengineering Institute's MPO assay kit. Reaction kinetics were dynamically monitored at 460 nm using a tetramethylbenzidine (TMB)-H₂O₂ colorimetric system. MPO activity was defined as the amount of enzyme catalyzing the conversion of 1 μmol of substrate per minute (U = 1 μmol / min). Simultaneously, total protein concentration was determined using the biuret-enhanced assay (BCA method). The final MPO activity data were standardized to total protein and expressed as per milligram of protein (U / mg protein) to eliminate the influence of differences in tissue sample size on the analytical results. In addition, a colorimetric method was used to determine the activity of β-N-acetylglucosidase (NAG) in the homogenate. This method quantifies enzyme activity by detecting the chromogenic changes produced after NAG catalyzes the hydrolysis of the corresponding substrate.

[0024] Analysis of mRNA expression of intestinal inflammation-related genes: Total RNA was extracted from the midgut using Trizol reagent. RNA quality was assessed by 1.0% agarose gel electrophoresis, and yield was determined using a NanoDrop ND-1000 spectrophotometer. After DNA removal using deoxyribonuclease, RNA was reverse transcribed into cDNA using the Revert Aid First Strand cDNA Synthesis kit according to the manufacturer's instructions. The cDNA template was stored at -20°C for further analysis. Primers were designed based on relevant sequences from NCBI and validated before quantitative PCR. Real-time quantitative PCR was performed using a quantitative thermal cycler. The amplification system consisted of 20 μL of 10 μL Power SYBR® Green PCR reaction mixture, 0.8 μL of forward and reverse primers (10 μ / mol), 6.4 μL of nuclease-free water, and 1 μL of cDNA template. The real-time fluorescence quantitative PCR program was as follows: 95℃ for 30 s, for a total of 40 cycles; 95℃ for 5 s, 60℃ for 20 s, and 65℃ for 15 s. Melting curve analysis was performed after the cycle. The internal reference gene was β-actin. Each detection gene was measured three times in duplicate, and the expression level of the target gene was calculated using the 2-ΔΔCt method.

[0025] High-throughput sequencing analysis of gut microbial diversity: hindgut samples were collected for gut microbiota community analysis. Total microbial genomic DNA was extracted using the EZNA® Soil DNA Kit, and DNA quality and concentration were detected by 1.0% agarose gel electrophoresis and a NanoDrop 2000 spectrophotometer. The V3-V4 region of the 16S rRNA gene was amplified by PCR using specific barcode primers (341F: 5'-ACTCCTACGGGAGGCAGCAG-3'; 806R: 5'-GGACTACHVGGGTWTCTAAT-3'). The resulting PCR products were analyzed by paired-end sequencing on an Illumina MiSeq PE 250 platform. The raw sequencing data were quality filtered using QIIME 1.7, followed by paired-end sequence assembly using FLASH 1.2. Operational taxonomic units (OTUs) were generated using Uparse based on a 97% similarity threshold. Taxonomic analysis of the bacterial community at the phylum and genus levels was performed for each sample. Based on OTU data, α-diversity indices (including Chao1, Shannon, and Simpson indices) and β-diversity indices (such as principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS)) were calculated. Furthermore, linear discriminant analysis of effect size (LEfSe) (LDA > 3, P < 0.05) was used to analyze significant differences in microbial composition at the genus level.

[0026] III. Results 1. Effects of bile acids on the growth performance of DSS-induced spotted scorpion. like Figure 1 As shown, compared with the control group, the experimental group without bile acid supplementation (DSSBA0) had significantly lower final body weight (FBW) and weight gain (WG) (P < 0.05). However, with the increase of bile acid supplementation level in the diet, growth performance showed a dose-dependent improvement trend, with the FBW and WG of the DSSBA30, DSSBA45, and DSSBA60 groups being significantly higher than those of the DSSBA0 and DSSBA15 groups (P < 0.05).

[0027] 2. Effects of bile acids on the intestinal index of DSS-induced stellate lentigines.

[0028] like Figure 2 As shown, the relative intestinal length (RIL), relative intestinal weight (RIW), and Zihler's index (ZI) of the DSSBA0 group were significantly lower than those of other treatment groups (P < 0.05), while the DSSBA60 group had the highest relative intestinal length, relative intestinal weight, and Zihler's index (P < 0.05). There were no significant differences between the control group and the DSSBA15 or DSSBA30 groups (P > 0.05).

[0029] 3. Effects of bile acids on intestinal morphology of DSS-induced spotted stellate foetida Figure 3 As shown, the submucosa of the control group fish was well-developed, with a thick muscle layer and intact mucosal structure. In contrast, the intestinal structure of the DSSBA0 group was further deteriorated, with mild atrophy of villi folds, and villi height and width significantly lower than other treatment groups (P < 0.05). Figure 4 A, 4B); Simultaneously, rupture of the mucosal fold tips was observed, and the muscle layer thickness was also significantly reduced (P < 0.05). Figure 4 (C). However, the addition of bile acids to the feed can alleviate the pathological effects of DSS on intestinal villi, especially in the high-dose bile acid group where intestinal structure was significantly improved. Quantitative analysis showed that, compared with the DSSBA0 group, the bile acid supplementation group had significantly increased villus height, width, muscle layer thickness, and goblet cell number.

[0030] 4. Effects of bile acids on the activity of intestinal inflammation-related enzymes in DSS-induced spotted stellate lentigines like Figure 5 As shown, the activities of myeloperoxidase (MPO) and β-N-acetyl-D-glucosidase (NAG) in the intestinal tissue of the DSSBA0 group were significantly higher than those of other treatment groups (P < 0.05). The intestinal MPO activity of the DSSBA60 group was significantly lower than that of the DSSBA15, DSSBA30 and control groups (P < 0.05).

[0031] 5. Effects of bile acids on the expression of intestinal inflammatory cytokine genes induced by DSS in East Star Spot like Figure 6As shown in the figure. Compared with the DSSBA0, DSSBA15, and DSSBA30 groups, the expression levels of TNF-α and IFN-γ mRNA in the control, DSSBA45, and DSSBA60 groups were significantly decreased (P < 0.05). The expression levels of IL-1β mRNA in the DSSBA60, DSSBA45, and control groups were significantly lower than those in the DSSBA0 and DSSBA15 groups (P < 0.05). IL-6 mRNA expression analysis showed that the expression level in the DSSBA60 group was significantly lower than that in the other treatment groups (P < 0.05), while the expression level in the DSSBA0 group was significantly higher than that in the other groups (P < 0.05). There were no significant differences among the Control, DSSBA15, DSSBA30, and DSSBA45 groups (P < 0.05). IL-8 mRNA expression analysis showed that the mRNA levels in the Control, DSSBA45, and DSSBA60 groups were significantly lower than those in other treatment groups (P < 0.05), with the expression level in the DSSBA0 group being significantly higher than that in other groups (P < 0.05). Compared with the DSSBA0, DSSBA15, and DSSBA30 groups, the expression of IL-10 gene in the Control, DSSBA45, and DSSBA60 groups was significantly upregulated (P < 0.05).

[0032] 6. Effects of bile acids on the expression of tight junction protein genes in the gut of DSS-induced spotted stellate foetida.

[0033] like Figure 7 As shown in the figure, compared with the control group, the expression levels of intestinal tight junction protein genes (ZO1, ZO2, ZO3, Occludin, Claudin-1, and MUC2) in the DSSBA0 group were significantly downregulated (P < 0.05), but the expression of these genes showed a significant upregulation trend with the increase of bile acid content in the diet. Specifically, the expression levels of ZO1, Occludin, Claudin-1, and MUC2 genes in the DSSBA60 group were significantly higher than those in other treatment groups (P < 0.05). The highest expression level of ZO2 gene was observed in the control group and the DSSBA60 group. In addition, the expression levels of ZO1 and ZO2 mRNA in the DSSBA0 and DSSBA30 groups were significantly lower than those in the control, DSSBA45, and DSSBA60 groups (P < 0.05). For the ZO3, Occludin, Claudin-1, and MUC2 genes, the expression levels in the DSSBA0, DSSBA15, and DSSBA30 groups were significantly lower than those in other treatment groups (P < 0.05).

[0034] 7. Effects of bile acids on the diversity and composition of the gut microbiota in DSS-induced spotted stellate foetida. Sequencing analysis was performed on intestinal samples from three groups: control, DSSBA0, and DSSBA60, yielding a total of 1,189,922 valid sequences. OTU (operational taxonomic unit) clustering analysis was performed at a 97% similarity level, identifying 5,674 OTUs. Venn diagrams showed that the control, DSSBA0, and DSSBA60 groups contained 557, 735, and 616 specific OTUs, respectively, with 115 (control vs. DSSBA0), 115 (control vs. DSSBA60), and 127 (DSSBA0 vs. DSSBA60) OTUs shared among the three groups, respectively. Figure 8 A). For example Figure 8 As shown in Figure B, in the Alpha diversity analysis, there were no significant differences in the Chao1 index, Ace index, Shannon index, and Simpson index (species diversity indicators) among the groups (P > 0.05). Principal coordinate analysis (PCoA) results showed that no obvious clustering was formed among the samples, indicating that there was no significant difference in Beta diversity of the microbial community among the three groups.

[0035] Phylum-level analysis showed that the dominant phyla (mean relative abundance > 1%) in the three groups were Proteobacteria (68.51%), Firmicutes (18.68%), Actinobacteriota (4.12%), Acidobacteriota (2.49%), Bacteroidota (1.38%), and Chloroflexi (1.18%). Compared with the DSSBA0 group, the abundance of Firmicutes was significantly increased in the DSSBA60 group (P < 0.05). Genus-level analysis showed that the dominant genera were *Achromobacter* (61.86%), *Candidatus_Arthromitus* (7.61%), *Lactobacillus* (4.50%), *Enterococcus* (1.20%), and *Enterobacter* (0.99%). Compared with the DSSBA0 group, the abundance of *Candidatus_Arthromitus*, *Enterococcus*, *Escherichia-Shigella*, and *Prevotella* was significantly increased in the DSSBA60 group (P < 0.05). LEfSe analysis further indicated that *Candidatus_Arthromitus* in the DSSBA60 group had a higher LDA value (LDA > 4), while no significantly different biomarkers (LDA > 4) were detected in the control group and the DSSBA0 group.

[0036] Microbial function analysis predicted by PICRUSt2 showed that the gut microbiota mainly participates in metabolic pathways, including basal metabolic pathways, secondary metabolite biosynthesis, multi-environment microbial metabolism, amino acid biosynthesis, and carbon metabolism. However, there were no significant differences in the relative abundance of the above metabolic pathways among the three groups (P > 0.05).

[0037] This invention intervened in DSS-induced enteritis in *Stachys pubescens* by adding different levels of bile acids (0.15%-0.60%). The results showed that adding 0.60% bile acids had the best effect on improving gut health. This study reveals the potential role of bile acids in alleviating DSS-induced enteritis and improving gut health in *Stachys pubescens*. The results indicated that bile acids significantly improved intestinal morphology, increasing villus height, enhancing muscular layer thickness, and improving overall structural integrity. These improvements were closely related to the upregulation of tight junction proteins (such as ZO-1 and Claudin-1), which play a crucial role in maintaining intestinal barrier function and regulating permeability. Furthermore, bile acids also alleviated intestinal inflammation by downregulating the expression of pro-inflammatory cytokines (such as TNF-α and IL-1β) and upregulating the expression of anti-inflammatory cytokines (such as IL-10). Simultaneously, bile acids also regulated the composition of the gut microbiota, increasing the proportion of beneficial Firmicutes and inhibiting the excessive proliferation of harmful Proteobacteria. Therefore, as a feed additive, bile acids have the potential to promote gut health in aquaculture and can become an effective strategy for improving fish health by reducing intestinal lesions and improving growth performance.

[0038] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for evaluating the role of bile acids in alleviating intestinal inflammation in spotted anthracis, characterized in that, Includes the following steps: S1. Inducing intestinal inflammation: A basic feed was formulated using fish meal, chicken meal, soy protein concentrate, flour, squid paste, fish oil, soybean oil, multivitamins, minerals and cellulose as raw materials. Chemical inducers were added to the basic feed to induce typical enteritis symptoms and construct an intestinal inflammation model of juvenile grouper. S2. Formulating bile acid feed: Add different levels of bile acids to the basic feed to formulate bile acid feed; S3. Feeding method: Juvenile East Star Grouper with enteritis symptoms were used as the experimental group and fed with feed containing bile acids. Healthy juvenile East Star Grouper were used as the control group and fed with basic feed without added bile acids. The feeding period was four weeks, and the fish were fed twice a day until they were full. S4. After feeding, all experimental fish were fasted for 24 hours. The number of fish in each net cage was counted and weighed. The weight gain rate was calculated. Six fish were randomly selected from each net cage and anesthetized with 50 mg / L tricaine mesylate. Their body weight, intestinal weight and length were measured. The relative intestinal weight, relative intestinal length and Zihler index were calculated. S5. Sampling Analysis: Histopathological observation of intestinal tissue: Midgut samples were taken from 3 fish, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned and stained with hematoxylin and eosin to observe intestinal morphological indicators. Inflammation-related enzyme activity detection: Midgut samples from 3 fish were collected, stored in liquid nitrogen, and tissue homogenates were prepared to detect myeloperoxidase and β-N-acetylglucosidase activities. Analysis of inflammation-related gene expression: Total RNA was extracted from midgut samples preserved in liquid nitrogen, reverse transcribed into cDNA, and the mRNA expression levels of pro-inflammatory and anti-inflammatory factors were measured by real-time quantitative PCR. Gut microbial diversity analysis: Hippu samples were taken from 3 fish, flash-frozen in liquid nitrogen, and microbial genomic DNA was extracted. The structure and diversity of the microbial community were analyzed by high-throughput sequencing of the V3-V4 region of the 16S rRNA gene.

2. The evaluation method for bile acids in alleviating intestinal inflammation in spotted prickly ... The chemical inducer is 0.75% sodium dextran sulfate.

3. The evaluation method for bile acids in alleviating intestinal inflammation in spotted prickly ... The different levels of bile acids include an addition amount of 0.15%-0.60% of the basal feed mass, and the total bile acid mass contains ≥68% porcine deoxycholic acid, ≥17% chenodeoxycholic acid and ≥9% porcine cholic acid.

4. The evaluation method for bile acids in alleviating intestinal inflammation in spotted prickly ... The bile acids alleviate intestinal inflammation through the following pathways: Upregulates the expression of tight junction protein genes, enhancing intestinal barrier function; It inhibits the mRNA expression of pro-inflammatory factors TNF-α and IL-1β, and promotes the expression of anti-inflammatory factor IL-10; It regulates the structure of the gut microbiota, enriches Firmicutes and inhibits Proteobacteria to optimize the gut microbiota structure.

5. The evaluation method for bile acids in alleviating intestinal inflammation in spotted prickly ... The bile acids can improve intestinal morphology indicators of the spotted dinoflagellate, including increasing the height of small intestinal villi, the thickness of the muscular layer, and the number of goblet cells.

6. The evaluation method for bile acids in alleviating intestinal inflammation in spotted anthracis according to claim 1, characterized in that, The bile acids reduce the activity of intestinal inflammation-related enzymes, including myeloperoxidase and β-N-acetylglucosidase.

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