Method for relieving perch liver injury and glucose metabolism disorder induced by high starch

Abstract

The invention relates to the technical field of biology, in particular to a method for relieving high-starch-induced liver injury and glucose metabolism disorder of perches. The feed formula provided by the invention comprises the following components in percentage by weight: 20-22% of cassava starch, 2-6% of mulberry leaf polysaccharide and the like. The mulberry leaf polysaccharide and high-content starch in the feed form efficient synergy, so that the growth performance of the weever is remarkably improved, accumulation of liver glycogen and fat is effectively reduced, and the health condition of the liver is remarkably improved; meanwhile, the blood sugar regulation capability of the weever is also optimized, the insulin resistance caused by high starch diet is reduced, and the insulin sensitivity is enhanced; and by activating a PI3K / Akt signal path and other mechanisms, a glucose metabolism path is optimized, and efficient utilization of energy is promoted. The feed disclosed by the invention is economical and efficient in formula, provides solid scientific support for healthy culture of perches, and powerfully promotes sustainable development of aquaculture industry.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for alleviating high-starch-induced liver injury and glucose metabolism disorder in Japanese sea bass. Background Art

[0002] In the field of aquaculture, Japanese sea bass has attracted much attention due to its high economic value and rich nutritional value. However, with the continuous increase in the cost of animal protein raw materials such as fish meal, the proportion of starch in the feed formula has been increasing. For example, the proportion of common corn flour is as high as 20% - 28% to reduce production costs. Although carbohydrates have economic advantages as an energy source, Japanese sea bass, as a carnivorous fish, has relatively limited adaptability of its digestive system to high-starch feeds. When the starch content in the feed exceeds a certain threshold (usually 9% - 15%), the growth performance and health status of Japanese sea bass will be significantly negatively affected. Long-term intake of high-starch feed will lead to imbalance of sugar metabolism homeostasis and abnormal accumulation of hepatic glycogen in Japanese sea bass, and then cause sugar metabolism disorder. This metabolic disorder is specifically manifested as hepatic steatosis, reduced antioxidant enzyme activity, and increased expression level of inflammatory factors. These pathological changes not only seriously damage the health status of Japanese sea bass, but also lead to a slowdown in its growth rate and a decline in aquaculture efficiency. When the starch level in the feed exceeds 15%, the weight gain rate of Japanese sea bass can be reduced by 23% - 35%, and at the same time, the feed coefficient increases by 0.3 - 0.5, further increasing the aquaculture cost.

[0003] To address the problems of high-starch-feed-induced liver injury and glucose metabolism disorder in Japanese sea bass, the main current improvement measures include physical process optimization, enzyme preparation addition, and nutritional balance regulation. Physical process optimization mainly improves the digestibility of the feed through low-temperature extrusion process (≤85°C) and the application of high-temperature-resistant enzyme preparations, but such process improvements are often accompanied by an increase in feed cost. Enzyme preparation addition aims to improve sugar metabolism efficiency by adding compound enzyme preparations. However, the activity of enzyme preparations is easily affected by processing temperature, and the effective action time in the intestine is limited, thus restricting its improvement effect. Nutritional balance regulation uses a feed formula with high protein and low starch, but this scheme significantly increases the feed cost and is not conducive to cost control in the aquaculture industry.

[0004] In recent years, research has begun to attempt to introduce natural active ingredients, such as plant extracts, in order to improve the adaptability of perch to high-starch diets. However, these solutions still face many technical challenges. First, most studies only use a single plant extract, with limited synergistic regulation of glycolipid metabolism. Second, some active ingredients are prone to degradation during the extrusion process, resulting in a significant reduction in their effective content in the feed. Finally, existing research lacks an in-depth exploration of the specific liver injury repair mechanism. Therefore, there is an urgent need to develop a new, efficient, and economical feed solution to improve the adaptability of perch to high-starch diets, enhance their growth performance and health status, while reducing breeding costs and promoting the sustainable development of the aquaculture industry. Summary of the Invention

[0005] In view of this, the present invention proposes a method for alleviating high-starch-induced liver injury and glucose metabolism disorders in perch.

[0006] The technical solution of the present invention is realized as follows:

[0007] In the first aspect, the present invention provides a feed for alleviating high-starch-induced liver injury and glucose metabolism disorders in perch, comprising the following components in weight percentages: fish meal 33%-37%, soy protein concentrate 21%-24%, fish oil 3.5%-4.5%, sodium alginate 0.8%-1.2%, carboxymethyl cellulose 0.8%-1.2%, soy lecithin 0.8%-1.2%, choline chloride 0.15%-0.25%, vitamin C 0.15%-0.25%, calcium dihydrogen phosphate 1.8%-2.2%, mineral premix 0.8%-1.2%, tapioca starch 20%-22%, and mulberry leaf polysaccharide 2%-6%, with the balance being microcrystalline cellulose;

[0008] The mineral premix comprises the following components in weight percentages: calcium gluconate 5%, ferrous sulfate 2%, magnesium sulfate 10%, sodium dihydrogen phosphate 10%, sodium chloride 2%, aluminum chloride 0.06%, potassium iodate 0.06%, potassium chloride 4%, copper sulfate 0.2%, manganese sulfate 0.4%, zinc sulfate 2%, and cobalt chloride 0.2%, with the balance being microcrystalline cellulose. Further, the weight percentage of mulberry leaf polysaccharide in the feed is 4%.

[0009] In the second aspect, the present invention provides the application of the feed in alleviating high-starch-induced liver injury and glucose metabolism disorders in perch, and the application is not for disease treatment.

[0010] Thirdly, the present invention provides a method for alleviating high-starch-induced liver injury and glucose metabolism disorder in Japanese seabass, which comprises the following steps: when Japanese seabass grows into the fingerling stage and its body weight reaches 60-80 grams, feed the fish with the above-mentioned feed; the total daily feeding amount of the feed is 4% of the total body weight of the fish, and feed the fish manually three times at fixed times every day: 8:30, 11:30 and 16:30, and the feeding cycle lasts for 8 weeks.

[0011] Fourthly, the present invention provides the application of the above method in alleviating high-starch-induced liver injury and glucose metabolism disorder in Japanese seabass, and this application is not for disease treatment.

[0012] The beneficial effects of the present invention at least include the following:

[0013] Firstly, by adding 2%-6% (preferably 4%) of mulberry leaf polysaccharide by mass ratio to the high-starch Japanese seabass feed, the growth performance of Japanese seabass is significantly improved, including the average body weight growth rate and the specific growth rate. This improvement is closely related to the role of mulberry leaf polysaccharide in improving liver health and sugar metabolism. Mulberry leaf polysaccharide can effectively reduce the accumulation of glycogen and fat in the liver, reduce the hepatosomatic index, and alleviate the pathological symptoms of liver cells caused by high-starch diet, such as cell swelling, vacuolization and nuclear disappearance.

[0014] Secondly, the addition of mulberry leaf polysaccharide also helps to regulate the blood glucose level of Japanese seabass. Long-term high-starch diet will cause the blood glucose of Japanese seabass to rise, insulin resistance to increase and sensitivity to decrease. While mulberry leaf polysaccharide can significantly improve the glucose uptake ability of liver cells, reduce insulin resistance and improve insulin sensitivity. This effect may be achieved by accelerating the repair and proliferation of pancreatic islet β cells, promoting insulin secretion, and through active transport and glycogen synthesis mechanisms.

[0015] In addition, mulberry leaf polysaccharide can also reduce the inflammatory response caused by high-starch diet and protect liver function. It can effectively reduce the abnormally elevated trypsin activity and reduce the risk of systemic inflammation, thereby maintaining the healthy state of the fish body.

[0016] Finally, mulberry leaf polysaccharide can also optimize the glucose metabolism mechanism of Japanese seabass. It may activate the PI3K / Akt signaling pathway, promote the binding of insulin to the insulin receptor on the cell surface, and enhance the uptake and transport of glucose by cells. At the same time, mulberry leaf polysaccharide can also up-regulate the activity of key glycolytic enzymes and inhibit the activity of key rate-limiting enzymes of gluconeogenesis, optimize the metabolic pathway of glucose in the liver, release energy, and promote the progress of other metabolic pathways.

[0017] In summary, the method of adding mulberry leaf polysaccharide to the high-starch Japanese seabass feed provided by the present invention has multiple beneficial effects such as promoting growth, improving liver health, regulating blood glucose, reducing inflammatory response and protecting liver function, providing strong support for the healthy aquaculture of Japanese seabass. Description of the Drawings

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0019] Figure 1 For the histological observation results of hematoxylin-eosin (HE) staining of the effect of mulberry leaf polysaccharide on the liver of perch fed a high-starch diet in the embodiments of the present invention, A-F respectively correspond to different treatment groups of LS0, HSM0, HSM2, HSM4, HSM6, and HSM8, with n = 3 in each group; the black arrow indicates cell enlargement, the red arrow indicates cell vacuolization, and the gray arrow indicates nuclear migration;

[0020] Figure 2 For the RNA-seq analysis of the HSM0 and HSM4 groups of perch liver tissues in the embodiments of the present invention; where: Figure 2 A is the principal component analysis (PCA) plot of the samples; Figure 2 B is the MA plot of the differential genes; Figure 2 C is the heatmap of different gene clusters; Figure 2 D is the sankey plot and bubble plot linked by pathway names (showing the enrichment factor value of the pathway through the bubble position, the number of genes enriched in the pathway through the bubble size, and the P value of the pathway through the bubble color); Figure 2 E is the gene set enrichment analysis (GSEA) based on differentially expressed genes (DEGs);

[0021] Figure 3 For the RNA-seq analysis and verification results of perch in the HSM0 and HSM4 groups in the embodiments of the present invention (n = 3); where: Figure 3 A is the Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation analysis; Figure 3 B is the KEGG functional enrichment analysis, which is the KEGG pathway analysis of differentially expressed genes in the liver when comparing the HSM4 group with the HSM0 group; Figure 3 C is the enrichment chords;

[0022] Figure 4 For the effect of mulberry leaf polysaccharide on the mRNA expression levels related to glucose metabolism in the liver of perch fed a high-starch diet in the embodiments of the present invention; where: Figure 4 A-C are the expression changes of key glycolysis genes; Figure 4 D is the expression of sugar metabolism transporter genes; Figure 4 E-F are the expressions of key gluconeogenesis genes;Figure 4 G-H represents the expression of pentose phosphate pathway genes; Figure 4 I-K represents the expression of genes related to insulin signal transduction; Different letters (a, b, c, d) indicate statistically significant differences between groups (P<0.05). Detailed implementation manners

[0023] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts belong to the scope of protection of the present invention. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0024]

Term Explanation

[0025] In some specific embodiments of the present invention, the meanings of relevant terms are as follows:

[0026] Table 1

[0027]

[0028]

[0029] I. Materials and Methods

[0030] 1. Animal Ethics Statement

[0031] All relevant operations of the animal experiments in this application comply with the regulations of the "Guide for the Care and Use of Laboratory Animals in China" (No. 2020-AFFRI-CAAS-001) in China, and are carried out with the approval of the Animal Experiment Ethics Committee of Changsha University; all experimental procedures comply with the requirements of the ARRIVE guidelines (Animal Research: Reporting of InVivo Experiments).

[0032] 2. Feed Preparation and Animal Rearing

[0033] Experimental fish of Micropterus salmoides were obtained from the Hunan Fisheries Science Research Institute in Changsha, China. A total of 540 healthy-looking bass (initial weight of about 70 grams) were randomly assigned to six treatment groups, with three replicates in each group.

[0034] Six kinds of isonitrogenous and isoenergetic feeds (LSM0, HSM0, HSM2, HSM4, HSM6, and HSM8) were prepared (the composition is shown in Table 2). After a two-week outdoor cage adaptation period, an eight-week formal aquaculture experiment was carried out.

[0035] As shown in Table 2, the components of the control group (LS0) are as follows:

[0036] A. Cassava starch content: 12% (mass ratio)

[0037] B. Mulberry leaf polysaccharide content: 0%

[0038] C. The proportion of other components is kept consistent with the HSM group, and the total weight is leveled by microcrystalline cellulose and bentonite (inert components). As shown in Table 2, the components of the high-starch group (HSM series) are as follows:

[0039] A. Unified cassava starch content: 21%

[0040] B. Mulberry leaf polysaccharide content: HSM0 (0%), HSM2 (2%), HSM4 (4%), HSM6 (6%), HSM8 (8%)

[0041] C. Except for the above components, the proportion of the remaining components is the same as that of the LS0 group, and the total weight is leveled by microcrystalline cellulose and bentonite.

[0042] Note: All contents are in mass ratio.

[0043] Table 2

[0044] Component (%) LS0 HSM0 HSM2 HSM4 HSM6 HSM8 Fish meal 35.00 35.00 35.00 35.00 35.00 35.00 Soybean protein concentrate 22.50 22.50 22.50 22.50 22.50 22.50 Fish oil 4.00 4.00 4.00 4.00 4.00 4.00 Soybean oil 3.00 3.00 3.00 3.00 3.00 3.00 Sodium alginate 1.00 1.00 1.00 1.00 1.00 1.00 Carboxymethyl cellulose 1.00 1.00 1.00 1.00 1.00 1.00 Soybean lecithin 1.00 1.00 1.00 1.00 1.00 1.00 Choline chloride 0.20 0.20 0.20 0.20 0.20 0.20 Vitamin C 0.20 0.20 0.20 0.20 0.20 0.20 Calcium dihydrogen phosphate 2.00 2.00 2.00 2.00 2.00 2.00 <![CDATA[Mineral premix (a) > 1.00 1.00 1.00 1.00 1.00 1.00 Cassava starch 12.00 21.0 21.0 21.0 21.0 21.0 Mulberry leaf polysaccharide 0.00 0.00 2.00 4.00 6.00 8.00 Microcrystalline cellulose 15.10 8.10 6.10 4.10 2.10 0.10 Bentonite 2.00 0.00 0.00 0.00 0.00 0.00

[0045] Note: (a) Each 1 kg of mineral premix contains the following components (by weight percentage): calcium gluconate 5%, ferrous sulfate 2%, magnesium sulfate 10%, sodium dihydrogen phosphate 10%, sodium chloride 2%, aluminum chloride 0.06%, potassium iodate 0.06%, potassium chloride 4%, copper sulfate 0.2%, manganese sulfate 0.4%, zinc sulfate 2%, cobalt chloride 0.2%. After all components are mixed, they are diluted to 1 kg with microcrystalline cellulose.

[0046] 3. Feed feeding

[0047] The feeding amount (mass ratio) of the experimental feed was 4% of the total weight of the fish, and the feeding amount was increased after the first 2 weeks. The daily feeding amount and conditions were recorded in a timely manner. The feeding period was 8 weeks. Before the experiment, the fish were disinfected and conditioned with conventional feed for 2 weeks to make them adapt to the experimental conditions and feed. The fish were fed manually three times a day at fixed times: 8:30, 11:30, and 16:30.

[0048] 4. Sample collection

[0049] After the 8-week feeding period ended, the fish were fasted for 24 h and then weighed and sampled. All sampled fish were anesthetized with MS-222 (Sigma-Aldrich, USA). The body weight, body length, and weights of visceral fat, viscera, and liver of each fish were measured. These data were used to calculate the weight gain rate (WGR), specific growth rate (SGR), hepatosomatic index (HSI), and viscerosomatic index (VSI) (as shown in Table 1).

[0050] Blood samples were drawn from the caudal vein and centrifuged at 4000×g for 10 min at 4 °C to obtain serum for hematological analysis. Liver samples were quickly excised, frozen in liquid nitrogen, and stored at -80 °C for subsequent experiments. In addition, four liver samples from each net of fish near the bile duct were fixed in tissue fixative (Sevier, China) for histological examination using HE staining.

[0051] 5. Feed analysis and evaluation of bioenzyme activity

[0052] First, the chemical composition of the feed was analyzed using the standard methods outlined in AOAC (2005), and the specific methods are as follows:

[0053] (1) Determination of moisture content (following the AOAC-925.10 method): The feed samples were dried to a constant weight in a DHG-9240A oven (Keelrein Instrument Co., Ltd., China) at 105 °C.

[0054] (2) Determination of crude protein content (following the AOAC-990.03 method): The Kjeldahl method (N×6.25) was used for determination, and the equipment used was a FOSS Kjeltec 2300 analyzer (Foss Analytical Instruments Co., Ltd., Sweden).

[0055] (3) Determination of crude fat content (following the AOAC-2003.05 method): The feed samples were extracted with ether in a Soxtec system without acid hydrolysis.

[0056] (4) Determination of ash content (following the AOAC-923.03 method): The feed samples were continuously incinerated in a muffle furnace (Shenyang Energy-Saving Electric Furnace Factory, China) at 550 °C for 12 h.

[0057] Subsequently, the glycogen levels in muscle and liver and the activities of multiple enzymes in the liver were evaluated in this application, and the methods are as follows:

[0058] (1) Glycogen level assessment: The glycogen levels in muscle and liver were accurately measured using a commercial kit (product number A043-1-1) provided by Nanjing Jiancheng Bioengineering Institute.

[0059] (2) Enzyme activity assessment: ELISA kits produced by Shanghai Enzyme-linked Biotechnology Co., Ltd. were used to assess the activities of enzymes such as hexokinase (HK), glucokinase (GK), phosphofructokinase (PFK), pyruvate kinase (PK), pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-bisphosphatase (FBPase), and glucose-6-phosphatase (G6Pase) in the liver.

[0060] 6. RNA extraction, cDNA library construction, and Illumina sequencing

[0061] Total RNA was extracted from tissues using the RNAsimple Total RNA Kit (TianGen, China, catalog number: DP419) according to the manufacturer's protocol. The RNA quality was evaluated using a 5300 Bioanalyser (Agilent) and quantified on an ND-2000 (NanoDrop Technologies). Only high-quality RNA samples (OD 260 / 280 = 1.8–2.2, OD 260 / 230 ≥ 2.0, RIN ≥ 6.5, 28S:18S ≥ 1.0, > 1 μg) were used for library construction.

[0062] RNA purification, reverse transcription, library construction, and sequencing were performed by Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China) according to the Illumina sequencing system (San Diego, California) manual. Using 1 μg of total RNA, according to Stranded mRNA Prep, Ligation method was used to prepare RNA-seq transcriptome libraries. First, mRNA was isolated by polyA selection method with oligo(dT) beads, and then fragmented with fragmentation buffer. Subsequently, double-stranded cDNA was synthesized using SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, CA) and random hexamer primers (Illumina). According to Illumina's library construction protocol, the synthesized cDNA was subjected to end repair, phosphorylation, and "A" base addition. Size selection of 300 bp for cDNA target fragments was performed on 2% low-range ultra-pure agarose, and then PCR amplification was carried out for 15 PCR cycles using Phusion DNA polymerase (NEB). The quantified paired-end RNA-seq sequencing libraries were sequenced on a NovaSeq 6000 sequencer (2×150 bp read length).

[0063] 7. De-assembly and unigene annotation

[0064] Use fastp [1] to trim and perform quality control on the original paired-end reads. Align the clean reads to the reference genome separately using HISAT2 in the directional mode [2] under. Assemble the mapped reads of each sample using StringTie with a reference-based method.

[0065] [1] Chen S, Zhou Y, Chen Y, et al. Fastp: an ultra-fast all-in-one FASTQ preprocessor[J]. Bioinformatics, 2018, 34(17): i884 - i890. DOI: 10.1093 / bioinformatics / bty560.

[0066] [2] Kim D, Langmead B, Salzberg S L. HISAT: A fast spliced aligner with low memory requirements[J]. Nature Methods, 2015, 12(4). DOI: 10.1038 / nmeth.3317.

[0067] 8. Detection of gene expression related to carbohydrate metabolism

[0068] Specific primers were designed for genes related to carbohydrate metabolism, and real-time fluorescence quantitative PCR (qPCR) was used to detect their expression levels in different treatment groups. The total volume of fluorescence quantitative PCR amplification was 10.0 μL, including 1.0 μL cDNA, 0.4 μL forward and reverse primers, 5.0 μL SYBR Green Pro Taq HS Premix (AG11718, Accurate Biology, China), and 3.2 μL ddH2O. The PCR conditions were: pre-denaturation at 95 °C for 30 s, followed by 40 cycles: denaturation at 95 °C for 3 s, annealing at 60 °C for 25 s, and extension at 72 °C for 10 s. qPCR detection was performed on a real-time quantitative PCR detector (B515A, Thermo Fisher Scientific, USA). The comparative CT method (2 -ΔΔCt method) was used to determine the relative expression levels of differentially expressed genes.

[0069] The expression differences of related genes were compared between different groups such as the high-starch feeding group and the mulberry leaf polysaccharide-added group, and the effects of mulberry leaf polysaccharide on the expression of genes related to carbohydrate metabolism were analyzed. The PCR primers were as follows:

[0070] Sequence information table

[0071]

[0072]

[0073] 9. Statistical analysis

[0074] Data analysis was performed using Excel and SPSS 25.0 software. One-way ANOVA and Duncan's multiple comparison test were used to evaluate the differences. P < 0.05 indicated statistical significance, and P > 0.05 indicated no significant difference. The results were expressed as mean ± standard error (SE).

[0075] II. Results

[0076] 1. Growth performance and morphological parameters

[0077] Compared with the 10% low-starch feeding group, the weight gain rate and specific growth rate of the high-starch group increased (P > 0.05), while the hepatosomatic index (HSI) and condition factor (CF) increased significantly (P < 0.05). As the content of mulberry leaf polysaccharide in the high-starch diet increased, the average weight gain rate (WGR) and specific growth rate (SGR) decreased (P > 0.05). At 4% mulberry leaf polysaccharide (HSM4), the specific growth rate of perch decreased, and it was the lowest at 8% mulberry leaf polysaccharide, while the visceral somatic index (VSI) and condition factor (CF) increased.

[0078] Table 3

[0079]

[0080]

[0081] Note: WGR = weight gain rate; SGR = specific growth rate; HSI = hepatosomatic index; VSI = viscerosomatic index; CF = condition factor index.

[0082] As can be seen from Table 3: With the increase of mulberry leaf polysaccharide content, WGR and SGR first increase and then decrease, reaching a relatively high level in the HSM4 group. The HSI of the high-starch group (HSM0) is higher than that of the low-starch group (LS0). Appropriate amounts of mulberry leaf polysaccharide (such as in the HSM2, HSM4, and HSM6 groups) can reduce HSI, approaching the level of the low-starch group, but excessive amounts (HSM8 group) will cause a significant increase in HSI. There is no significant pattern in the viscerosomatic index among the experimental groups; the condition factor shows certain fluctuating changes among the experimental groups.

[0083] Therefore, compared with the 10% low-starch group, the HSM0 group significantly reduces the WGR and SGR of largemouth bass. With the increase of mulberry leaf polysaccharide content, WGR and SGR first increase and then decrease, reaching the highest level at 4% mulberry leaf polysaccharide (HSM4), indicating that appropriate amounts of mulberry leaf polysaccharide can improve the decline in growth performance caused by a high-starch diet, but excessive amounts have poor effects. The hepatosomatic index (HSI) of the high-starch group increases significantly. Appropriate amounts of mulberry leaf polysaccharide (such as 2% - 6%) can effectively offset the increase in HSI caused by high starch, reduce visceral fat, lower the condition factor index (CF), and restore the healthy state of the fish. It helps the fish recover to a healthier state; however, a high content of 8% mulberry leaf polysaccharide will significantly increase HSI, while there is no significant difference in VSI. Although there is no significant pattern in the viscerosomatic index (VSI) among the experimental groups, considering key indicators such as WGR, SGR, HSI, and condition factor index (CF), a 4% content of mulberry leaf polysaccharide can achieve a relatively good balance in terms of growth performance and liver health, approaching a relatively optimal state for each indicator.

[0084] 2. Histological examination

[0085] This application studies the effect of mulberry leaf polysaccharide on high-starch-induced liver damage in bass by observing the microstructure of the liver. As Figure 1As shown, A - F correspond to LS0, HSM0, HSM2, HSM4, HSM6, and HSM8 respectively. The cells in the LS0 experimental group maintained a normal morphology without significant enlargement, while most cells in the HSM0 group showed abnormal enlargement, vacuolization, and glycogen pushing the nucleus to the edge. This indicates that perch experienced pathological glycogen accumulation due to excessive intake of digestible starch. Although there were still some cell abnormalities in the HSM4 group, such as enlargement, vacuolization, and nuclear migration, these were closer to the cell states observed in LS. In contrast, the condition in the HSM8 group deteriorated, with a significant increase in nuclear abnormalities, and this level of mulberry leaf polysaccharide actually exacerbated the pathological glycogen accumulation in the liver.

[0086] 3. Detection of liver glycogen, muscle glycogen, and serum biochemical indicators

[0087] This application studies the physiological responses of perch under different starch - level diet treatments, especially the systematic determination and analysis of serum biochemical indicators, liver glycogen, and muscle glycogen content. The experimental design uses the low - starch diet group (LS0) as a control to deeply explore the physiological changes in the high - starch diet groups (HSM0 to HSM8) after adding mulberry leaf polysaccharide.

[0088] The experimental results show that compared with the low - starch diet group, the high - starch diet treatment significantly increased the glucose concentration, insulin level, and trypsin activity in the serum of perch. Especially under the high - starch diet condition, by adding 4% and 6% of mulberry leaf polysaccharide to the feed, the blood glucose can be effectively maintained within the normal range. In addition, the muscle insulin level of perch in the high - starch diet group was significantly higher than that in the low - starch group, and showed a trend of initially increasing and then decreasing with the increase in the addition amount of mulberry leaf polysaccharide, although this change did not show a unified pattern. It is worth noting that although the glucagon level in the high - starch diet group was slightly higher than that in the low - starch group (statistical significance P > 0.05), after the intervention of mulberry leaf polysaccharide, its level first increased significantly and then decreased (P < 0.05). The specific data are shown in Table 4.

[0089] Based on this analysis, combined with the dynamic changes of insulin and glucagon, it is speculated that mulberry leaf polysaccharide may participate in and affect the homeostasis regulation process of blood glucose by regulating the secretion and function of these two hormones. At the same time, mulberry leaf polysaccharide may also affect the digestive enzyme activity of perch, thereby regulating its digestive efficiency and nutrient absorption ability for food. However, different addition amounts of mulberry leaf polysaccharide show different effects in regulating glycogen reserves, blood glucose levels, etc. Generally speaking, the addition range of 2% to 6% performs better in most indicators.

[0090] Furthermore, compared to the low-starch control group, a high-starch diet led to a significant accumulation of liver glycogen and muscle glycogen in perch, suggesting a risk of hyperglycemia. The addition of mulberry leaf polysaccharide significantly reduced the liver glycogen content, restoring it to the normal physiological level. For muscle glycogen, its content first decreased significantly after the intervention of mulberry leaf polysaccharide and then increased significantly. Although the liver glycogen content showed a fluctuating trend of first decreasing and then increasing, overall, the addition of mulberry leaf polysaccharide prevented the liver glycogen from maintaining at the high level induced by the high-starch diet, but promoted its reduction and stabilization within a more reasonable range. These findings strongly demonstrated the effectiveness of mulberry leaf polysaccharide in reducing glycogen levels.

[0091] In summary, a high-starch diet has a profound impact on blood glucose homeostasis, insulin secretion, and glycogen metabolism in perch, while mulberry leaf polysaccharide exhibits a positive potential role in regulating these key physiological processes, especially in significantly reducing glycogen accumulation. Considering various physiological indexes comprehensively, the addition of 2% to 6% mulberry leaf polysaccharide shows good efficacy in regulating blood glucose, liver glycogen, and muscle glycogen levels.

[0092] Table 4

[0093]

[0094]

[0095] 4. Detection of enzyme activities related to glucose metabolism

[0096] To further explore how mulberry leaf polysaccharide affects blood glucose regulation through key enzymes in glucose metabolism, we evaluated the activities of these enzymes. As shown in Table 5, after an 8-week feeding experiment, the activities of liver glucose metabolism enzymes in perch were analyzed. Compared with the low-starch group, the activity of hexokinase (HK) in the high-starch group was higher (P < 0.05). In addition, the activities of pyruvate kinase (PK) and phosphofructokinase (PFK) in the high-starch group also increased, but not significantly (P > 0.05). The addition of mulberry leaf polysaccharide to the high-starch diet led to a significant decrease in HK activity, while the activities of PK and PFK initially increased significantly. The most significant regulatory effect occurred at the 4% supplementation level (HSM4) (P < 0.05). The activities of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) decreased with the increase in starch level and further decreased with the supplementation of mulberry leaf polysaccharide. The activity of fructose-1,6-bisphosphatase (FBP) was not significantly affected by different starch levels (P > 0.05), but increased significantly through the supplementation of 6% and 8% mulberry leaf polysaccharide (P < 0.05).

[0097] Based on the above data analysis, generally speaking, adding 4% to 6% of mulberry leaf polysaccharide showed relatively superior performance in most evaluation indicators. Within this addition range, the activity of HK decreased significantly, effectively avoiding the problem of abnormal increase in the activity of this enzyme caused by high-starch diet; at the same time, the activities of PK and PFK increased significantly, strongly promoting the process of glycolysis. In addition, the activities of PEPCK and G6Pase decreased further, contributing to reducing gluconeogenesis; while the activity of FBP increased significantly, playing a positive regulatory role in maintaining glucose metabolism balance. Compared with other addition levels, 4% to 6% of mulberry leaf polysaccharide showed more significant performance in regulating the activities of enzymes closely related to glucose metabolism and could better maintain the stability of blood glucose level.

[0098] Table 5

[0099]

[0100]

[0101] 5. Illumina Sequencing and Functional Annotation

[0102] Statistical analysis of the sequencing reads showed that a total of 323,306,132 raw reads were generated. From these, 320,706,740 clean reads were obtained, accounting for 99.75% to 99.86% of the total reads of each sample. Among these clean reads, 73.17% to 78.21% were successfully mapped to the reference genome. The GC content of the clean reads ranged from 51.56% to 53.55%. In addition, the Q20 values of the cDNA libraries of all six groups exceeded 97%, and the Q30 values were higher than 93% (Table 6).

[0103] Table 6

[0104] Group Rawreads Cleanreads Q20(％) Q30(％) GC content (%) LSM0 62669230 62088324 97.91 96.13 48.76 HSM0 69736264 69177730 97.94 96.19 49.23 HSM2 45156550 44856226 97.64 93.17 48.20 HSM4 47222634 46724508 97.85 95.98 48.77 HSM6 46604470 46294710 97.73 93.31 48.38 HSM8 51916984 51565242 97.64 93.15 48.20

[0105] 6. Identification and Analysis of Differentially Expressed Genes (DEGs)

[0106] This analysis identified a total of 29,048 expressed genes, including 28,077 known genes and 971 new genes. The total number of expressed transcripts was 59,554, of which 44,738 were known transcripts and 14,816 were new transcripts. Using the quantitative expression level data, differential gene analysis was performed between groups, revealing different expression levels between the two groups. In the HSM4 and HSM0 groups, a total of 13,474 genes were co-expressed, and 598 and 532 genes were unique to each group respectively. This indicates that there are significant differences in the gene expression of the liver of perch under high-starch diet. Compared with the HSM0 group, the HSM4 group showed 823 genes with significantly different expression levels, including 472 up-regulated genes and 351 down-regulated genes (Figure 2 B). Further clustering analysis of these differentially expressed genes emphasized the impact of the high-starch diet on the gene expression profile of the liver of perch.

[0107] 7. Functional enrichment analysis of DEGs

[0108] The DEGs were annotated using the GO database and their functions were evaluated. GO analysis showed that the enrichment function distributions of DEGs were similar between the HSM4 and HSM0 groups (Table 7). All DEGs were classified into three main functional categories: cellular component, molecular function, and biological process. Specifically, 20 significantly enriched terms were classified as biological processes (BP), including cellular process, metabolic process, biological regulation, developmental process, cellular component organization or biogenesis, localization, response to stimulus, multicellular organismal process, etc. In addition, 14 significantly enriched terms were classified as molecular functions (MF), such as membrane part, cell part, organelle, organelle part, and protein-containing complex. The remaining 10 significantly enriched terms belonged to cellular components (CC), including binding, catalytic activity, transporter activity, molecular function regulator, and molecular transducer activity. (Table 7)

[0109] Table 7

[0110]

[0111]

[0112] Note: BP represents biological process; CC represents cellular component; MF represents molecular function. Only the top 8 BPs, top 4 CCs, and top 7 MFs are listed in this table.

[0113] The DEGs were further subjected to pathway enrichment analysis in the KEGG database. The analysis identified significant enrichment (P<0.05) of 50 signaling pathways, including pancreatic secretion, glycolysis / gluconeogenesis, starch and sucrose metabolism, neomycin biosynthesis, fat digestion and absorption, cysteine and methionine metabolism, carbohydrate digestion and absorption, glycine, serine and threonine metabolism, central carbon metabolism in cancer, etc. Figure 2 , Figure 3 B). The research results showed that compared with the HSM0 group, the gene expression patterns related to glucose metabolism and cell survival in the liver of perch in the HSM4 group changed significantly. Figure 3 A, 3C)

[0114] 8. Expression of genes related to carbohydrate metabolism

[0115] In high-starch-fed perch, the expression levels of key genes related to the gluconeogenesis pathway, such as pepck (phosphoenolpyruvate carboxykinase), showed a downward trend compared with the low-starch control group, although there was no significant difference ( Figure 4 E). However, as Figure 4 shown in D, after adding mulberry leaf polysaccharide, the expression levels of g6pase and glut2 were significantly up-regulated, while pepck was down-regulated (P>0.05). Compared with the HSM0 group, the relative expression levels of the key glycolytic genes hk, pfk, and pk in the HSM4 group increased (P<0.05) ( Figure 4 A-C). The expression levels of irs1, glp-1r, 6pgdh, and g6pdh initially increased significantly with the addition of mulberry leaf polysaccharide in the high-starch diet and then decreased significantly.

[0116] 9. Enrichment analysis of transcription factors

[0117] The results of the enrichment analysis of transcription factors are shown in Table 8. From the transcription factor data predicted by the transcriptome, multiple transcription factors were identified, such as lmx1bb, klf11a, etc. Among them, the klf11a gene appeared multiple times and had a low E value, indicating that this gene had a high credibility in the transcriptome prediction and might play an important role in the physiological processes regulated by mulberry leaf polysaccharide. The important role of the klf11a gene in the regulation of glucose metabolism by mulberry leaf polysaccharide. Mulberry leaf polysaccharide might activate specific transcription factors, such as klf11a, and then activate the PI3K / Akt signaling pathway, stimulate insulin secretion, regulate glucose metabolism, and ultimately achieve the effect of reducing blood sugar.

[0118] Table 8

[0119]

[0120]

[0121] III. Conclusion

[0122] This application first provides a method for improving the growth performance, reducing liver damage, and regulating blood sugar levels of perch by appropriately adding mulberry leaf polysaccharide to the feed under high-starch diet conditions. Specifically, this method includes adding mulberry leaf polysaccharide to the high-starch feed to achieve the following effects:

[0123] 1. Growth performance and improvement of liver health: Mulberry leaf polysaccharide at a mass ratio of 2%-6% in high-starch feed can significantly increase the average weight rate and specific growth rate of perch, while effectively reducing the accumulation of glycogen and fat in the liver and significantly decreasing the hepatosomatic index. Confirmed by liver histopathological examination, mulberry leaf polysaccharide can significantly alleviate pathological symptoms such as cell swelling, vacuolization and nuclear disappearance caused by high-starch diet. It is speculated that its mechanism of action is related to the reduction of blood lipid by mulberry leaf polysaccharide and the inhibition of glycogen and fat accumulation in hepatocytes.

[0124] 2. Blood glucose regulation and improvement of insulin sensitivity: Long-term high-starch diet can lead to persistent hyperglycemia in perch, manifested as increased serum glucose and insulin levels, decreased insulin resistance and sensitivity. Administration of 4% mulberry leaf polysaccharide can significantly improve the glucose uptake ability of hepatocytes. It is speculated that it promotes insulin secretion by accelerating the repair and proliferation of pancreatic islet β cells, and then through the mechanisms of active transport and glycogen synthesis, promotes serum glucose to enter hepatocytes, reduces the harmful effects of insulin resistance, and improves the insulin sensitivity of cells.

[0125] 3. Reduction of inflammatory response and protection of liver function: High-starch diet can exacerbate oxidative stress, inflammatory response and liver function impairment in perch. Mulberry leaf polysaccharide can effectively reduce the abnormally increased trypsin activity caused by high-starch intake, indicating that it can alleviate the inflammatory response caused by hyperglycemia stress to a certain extent, reduce the risk of systemic inflammation, and thus play a role in protecting liver function.

[0126] 4. Glucose metabolism regulation mechanism: Carnivorous fish have limited utilization efficiency of carbohydrates. The addition of mulberry leaf polysaccharide significantly affects blood glucose levels by regulating the expression of genes related to glucose metabolism. Transcriptome analysis shows that mulberry leaf polysaccharide may activate the PI3K / Akt signaling pathway, promote the binding of insulin to the IR (Insulin Receptor) on the cell surface, and then activate downstream signal transduction, affect glucose transporters, and enhance the uptake and transport of glucose by cells. In addition, mulberry leaf polysaccharide also optimizes the metabolic pathway of glucose in the liver by upregulating the activity of key glycolytic enzymes and inhibiting the activity of key rate-limiting enzymes of gluconeogenesis, converting it into liver glycogen and other non-glycogen substances, releasing energy, and promoting the progress of other metabolic pathways such as fat metabolism and amino acid metabolism. Combining the results of gene expression changes and transcription factor enrichment analysis shows that mulberry leaf polysaccharide may activate specific transcription factors such as klf11a, activate the PI3K / Akt signaling pathway, stimulate insulin secretion, and then regulate glucose metabolism to achieve the effect of reducing blood glucose.

[0127] The above is only the preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A feed for alleviating high-starch-induced liver injury and glucose metabolism disorder in perch, characterized in that, Comprising the following components in percentage by weight: fish meal 33% - 37%, soy protein concentrate 21% - 24%, fish oil 3.5% - 4.5%, sodium alginate 0.8% - 1.2%, carboxymethyl cellulose 0.8% - 1.2%, soy lecithin 0.8% - 1.2%, choline chloride 0.15% - 0.25%, vitamin C 0.15% - 0.25%, calcium dihydrogen phosphate 1.8% - 2.2%, mineral premix 0.8% - 1.2%, cassava starch 20% - 22% and mulberry leaf polysaccharide 2% - 6%, the balance being microcrystalline cellulose; The mineral premix comprises the following components in percentage by weight: calcium gluconate 5%, ferrous sulfate 2%, magnesium sulfate 10%, sodium dihydrogen phosphate 10%, sodium chloride 2%, aluminum chloride 0.06%, potassium iodate 0.06%, potassium chloride 4%, copper sulfate 0.2%, manganese sulfate 0.4%, zinc sulfate 2% and cobalt chloride 0.2%, the balance being microcrystalline cellulose.

2. The feed according to claim 1, wherein The weight percentage of mulberry leaf polysaccharide in the feed is 4%.

3. Use of the feed according to claim 1 or 2 in alleviating high-starch-induced liver injury and glucose metabolism disorder in perch, said use not being for disease treatment.

4. The application according to claim 3, characterized in that The use includes at least one of the following uses: (A1) Improving the problem of decreased growth performance caused by a high-starch diet, the indicators of the growth performance including average body weight growth rate and specific growth rate; (A2) Alleviating liver injury caused by a high-starch diet, which includes: alleviating the enlargement, vacuolization and nuclear migration of liver cells; alleviating the accumulation of glycogen and fat in the liver; and alleviating the increase in hepatosomatic index; (A3) By regulating insulin, glucagon, and controlling the activities of glucose metabolism enzymes, improving the problem of blood glucose homeostasis imbalance in perch caused by a high-starch diet, promoting the blood glucose level to tend to be stable and maintaining glucose metabolism balance; the glucose metabolism enzymes include hexokinase, pyruvate kinase, phosphofructokinase, phosphoenolpyruvate carboxykinase, glucose-6-phosphatase and fructose-1,6-bisphosphatase; (A4) Enhancing the ability of perch to metabolize and process glucose in a high-starch feed, reducing the risk of hyperglycemia, maintaining blood glucose at a normal level, improving the tolerance of perch to a high-starch feed, and at the same time enhancing the growth performance of perch to increase aquaculture production.

5. A method for alleviating high-starch-induced liver injury and glucose metabolism disorder in perch, characterized in that, When perch grow into the fingerling stage and their body weight reaches 60 - 80 grams, the feed according to claim 1 or 2 is used for feeding; the total daily feeding amount of the feed is 4% of the total body weight of the fish, and it is fed three times at fixed times every day, and the feeding cycle lasts for 8 weeks.

6. Use of the method according to claim 5 in alleviating high-starch-induced liver injury and glucose metabolism disorder in perch, said use not being for disease treatment.

7. The application according to claim 6, characterized in that, For improving the problem of decreased growth performance caused by a high-starch diet and alleviating liver injury caused by a high-starch diet; the indicators of the growth performance include average body weight growth rate and specific growth rate.

8. The application according to claim 7, wherein The liver injury caused by the high-starch diet includes at least one of the following: (1) the enlargement, vacuolization and nuclear migration of liver cells; (2) the accumulation of glycogen and fat in the liver; (3) the increase in hepatosomatic index.

9. The application according to claim 6, characterized in that, By regulating insulin, glucagon, and controlling the activities of glucose-metabolizing enzymes, the problem of blood glucose homeostasis imbalance in perch caused by a high-starch diet is improved, promoting the stabilization of blood glucose levels and maintaining glucose metabolism balance; the glucose-metabolizing enzymes include hexokinase, pyruvate kinase, phosphofructokinase, phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and fructose-1,6-bisphosphatase.

10. The application according to claim 6, characterized in that Enhance the perch's ability to metabolize and process glucose in high-starch feed, reduce the risk of hyperglycemia, maintain blood glucose at normal levels, improve the perch's tolerance to high-starch feed, and at the same time enhance the perch's growth performance to increase aquaculture production.

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