Application of taurine in preparing medicine for preventing gosling hyperuricemia nephropathy through target EGFR (Epidermal Growth Factor Receptor)

Abstract

The invention provides application of taurine in preparation of a medicine for preventing gosling hyperuricemia nephropathy through a target EGFR (epidermal growth factor receptor), and belongs to the technical field of gosling hyperuricemia. The taurine is proved to be capable of reducing UA generation, promoting UA excretion, relieving inflammatory response, resisting oxidative damage and delaying the renal tubulointerstitial fibrosis process, so that the taurine plays a role in protecting the kidney of the HN gosling.

Description

Technical Field

[0001] This invention relates to the field of gosling hyperuricemia technology, and more particularly to the application of taurine targeting EGFR in the preparation of drugs for the prevention of gosling hyperuricemia nephropathy. Background Technology

[0002] Hyperuricemic nephropathy (HN) is a common complication of hyperuricemia (HUA). HUA is a condition caused by an abnormally high level of uric acid (UA) in the body due to excessive production and / or reduced excretion. Currently, the morbidity and mortality rates of HN in goslings are extremely high in the poultry industry, causing serious economic losses. Therefore, it is essential to pay attention to and strengthen the prevention of HN.

[0003] The causes of hepatitis B (HN) are complex. First, in terms of the rearing environment: raising goslings in damp, dark sheds, intensive management, and insufficient exercise; second, in terms of feeding: unscientific feed formulation, excessive content of nucleoproteins, purine bases, and soluble calcium salts in the feed, excessive intake of purines and nucleic acid proteins increases uric acid (UA) levels, and insufficient water intake prevents the kidneys from excreting high levels of UA in a timely manner; other factors include: genetic factors, kidney metabolic dysfunction caused by certain microorganisms and viral infections, and other primary causes of hepatitis B leading to abnormal purine metabolism and reduced renal UA excretion.

[0004] Current prevention and control measures primarily focus on feed formulation, ensuring feed is scientifically and rationally formulated according to the needs of the organism's growth, development, and production. Environmental control measures include preventing feed spoilage, maintaining hygiene in the rearing environment, ensuring timely ventilation, providing ample drinking water, and promoting sufficient exercise. Drug-based prevention involves using medications that reduce uric acid (UA) production and promote UA excretion, such as xanthine oxidase inhibitors (alopurinol, febuxostat), recombinant uricase (raburicase), and uric acid excretion agents (probenecid). However, these drugs can damage the kidneys, so careful selection is crucial, especially avoiding long-term excessive use of sulfonamides. Finally, adding 2.5%–3.0% NaHCO3 to the feed can also promote urate excretion. Currently, there is no satisfactory treatment for geese already infected with hematuria (HN). Therefore, prevention is the primary focus for HN.

[0005] 2-Aminoethanesulfonic acid, abbreviated as taurine (Tau), with the molecular formula NH₂CH₂CH₂SO₃H, is a ubiquitous sulfur-containing amino acid derivative. It is a non-essential amino acid found in most animal tissues and plays a vital physiological role. Tau is a simple yet unique sulfur-containing amino acid with a wide range of biological functions and no toxic side effects.

[0006] Poultry lack uricase, making them unable to oxidize poorly soluble uric acid (UA), leading to elevated UA levels and a high risk of hemorrhagic nephritis (HN). Meanwhile, geese are herbivorous poultry with the ability to digest and absorb crude fiber. However, goose farmers, in their pursuit of improved growth performance, often blindly feed them broiler or duck feed, causing abnormal UA production and metabolism, resulting in HN. HN typically has a high morbidity and mortality rate, causing significant economic losses to the goose farming industry. Furthermore, currently used clinical drugs can damage the kidneys and gastrointestinal tract, causing irreversible harm. Therefore, finding safe and effective drugs and nutritional immune supplements to prevent HN is of significant scientific importance for goose farming.

[0007] Previous studies using a rat HN model have shown that tau can reduce UA production and promote UA excretion, thereby lowering serum UA levels and alleviating or reversing kidney damage. However, the specific effects and regulatory mechanisms of tau on goslings remain unclear and require further investigation.

[0008] Based on this, the present invention is proposed. Summary of the Invention

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of taurine targeting EGFR in the preparation of drugs for the prevention of hyperuricemic nephropathy in goslings.

[0010] Preferably, the taurine is added to the goslings' daily age.

[0011] Preferably, the amount of taurine added to the gosling diet is 0.05% to 0.15%.

[0012] This invention confirms that taurine can reduce UA production and promote UA excretion, thereby protecting the kidneys of HN goslings by alleviating inflammatory responses, resisting oxidative damage, and delaying the process of renal tubular interstitial fibrosis. Attached Figure Description

[0013] Figure 1 The effect of taurine on kidney and liver indices in goslings; Figure 2 Section of kidney tissue from goslings (HE, 400×). Figure 3 The effect of taurine on the levels of UA, BUN, and CRE in the serum of goslings; Figure 4 The effect of taurine on the levels of AST, ALT, and LDH in the serum of goslings; Figure 5 The effect of taurine on the levels of XOD and ADA in the serum of goslings; Figure 6 The effect of taurine on the XOD and ADA content in the liver of goslings; Figure 7 The effect of taurine on the relative expression levels of genes related to uric acid production in the liver of goslings; Figure 8 The effect of taurine on the relative expression levels of genes related to uric acid reabsorption in the kidneys of goslings; Figure 9 The effect of taurine on the relative expression levels of genes related to uric acid excretion in the kidneys of goslings; Figure 10 The effect of taurine on the levels of TNF-α, IL-1β, and MCP-1 in the kidneys of goslings; Figure 11 The effect of taurine on the levels of TNF-α, IL-1β, and MCP-1 in the kidneys of goslings; Figure 12 The effect of taurine on the relative expression levels of genes related to kidney inflammation in goslings; Figure 13 Effects of taurine on SOD and MDA levels in gosling serum Figure 14 The effect of taurine on the SOD and MDA content in the kidneys of goslings; Figure 15 The effect of taurine on the relative expression levels of antioxidant-related genes in the kidneys of goslings; Figure 16 The effect of taurine on the TGF-β content in the kidneys of goslings; Figure 17 The effect of taurine on the relative expression levels of genes related to renal tubulointerstitial fibrosis in goslings; Figure 18 The effects of taurine on the expression of COL1A1 and α-SMA, marker proteins of renal tubular interstitial fibrosis in goslings; Figure 19 Effect of taurine on COL1A1 protein expression in the kidneys of goslings (×400). Figure 20 Effect of taurine on α-SMA protein expression in gosling kidney tissue (×400); Figure 21 The effect of taurine on the phosphorylation level of EGFR in goslings. Detailed Implementation

[0014] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0015] Example 1

[0016] 1. Materials and Methods

[0017] 1.1 Test Materials

[0018] 1.1.1 Laboratory animals and their feeding and management

[0019] Sixty healthy 10-day-old goslings were provided by a breeding farm in Linyi City, Shandong Province. The trial period lasted 21 days, during which they had free access to feed and water.

[0020] 1.1.2 Experimental Diets

[0021] Table 1. Raw materials and nutrient composition of the experimental diets

[0022] Content per kg of premix: Vitamin A, 12000 IU; Vitamin D3, 1000 IU; Vitamin E, 20 IU; Vitamin K, 1.0 mg; Vitamin B1, 2.0 mg; Vitamin B2, 7.0 mg; Vitamin B6, 3.0 mg; Vitamin B12, 0.02 mg; Biotin, 0.20 mg; Folic acid, 0.6 mg; Calcium pantothenate, 10 mg; Niacin, 20 mg; Cu (CuSO4·5H2O), 25 mg; I (KI), 1 mg; Fe (FeSO4·7H2O), 80 mg; Mn (MnSO4·H2O), 120 mg; Se (NaSeO3), 0.3 mg; Zn (ZnO), 80 mg. (Calculated values)

[0023] 2.1.3 Experimental Equipment and Reagents

[0024] As shown in Tables 2 and 3.

[0025] Table 2 Test Instruments

[0026] Table 3 Test Reagents

[0027] 1.2 Test Methods

[0028] 1.2.1 Experimental Grouping and Treatment

[0029] Ten-day-old healthy calico goslings were randomly divided into four groups (equal in weight, sex, etc.): a normal control group (Group C), a hyperuricemia nephropathy model group (Group M), a taurine prevention group (Group MT), and a taurine control group (Group T). Group C was fed normal complete goose feed, Group M was fed high-protein, high-calcium goose feed, Group MT was fed feed supplemented with 0.1% taurine (Group M feed), and Group T was fed feed supplemented with 0.1% taurine (Group C feed). Each group consisted of 15 goslings, and the experiment lasted 21 days. During the feeding period, the goslings had free access to feed and water. Specific experimental groupings and feed formulations are shown in Table 1.

[0030] 1.2.2 Sample Collection

[0031] Blood was collected from the heart of 31-day-old goslings after a 12-hour fast. The blood was then placed at room temperature and tilted for half an hour before centrifugation at 3000 rpm for 15 minutes. The supernatant was collected and stored at -80°C for later use. After euthanizing the goslings, they were quickly dissected, and the left and right kidneys and livers of each group were removed. The right kidney and liver were weighed using an analytical balance, and the data were recorded. The left kidney was fixed in 4% paraformaldehyde for approximately 4 weeks, then embedded in paraffin to prepare paraffin sections for subsequent experiments. The right kidney and liver were minced and placed in cryovials, immediately frozen in liquid nitrogen, and then stored at -80°C for later use.

[0032] 1.2.3 Clinical observation of goslings and determination of kidney and liver indices

[0033] Observe the mental state, appetite and defecation of the experimental goslings every day.

[0034] The right kidney was harvested and weighed. The renal index was calculated using the nimodipine method, i.e., renal index = .

[0035] The liver is collected and weighed, and the calculation method is the same as for the kidneys, i.e., liver index = .

[0036] 1.2.4 Histological examination of gosling kidneys

[0037] 1.3.2.1 Sample Collection and Preparation

[0038] 1×1×0.5cm 3 The kidneys of goslings were placed in a specimen bottle containing 3 ml of 4% paraformaldehyde.

[0039] 1.3.2.2 Paraffin Embedding

[0040] Rinsing: Rinse the kidneys of the fixed goslings slowly with running water for 8 hours; Dehydration: Dehydrate the rinsed gosling kidneys by soaking them in 70%, 80%, 90%, and 95% alcohol solutions for 12 hours, and then soaking them in 100% alcohol for 10 minutes. Transparency: Absorb excess fluid from the surface of the gosling's kidneys, and place the sample into xylene I and II sequentially for about 2 minutes to achieve tissue transparency; Wax infiltration: Immerse the clear kidney tissue of goslings in melted paraffin for about 3 hours until the wax infiltration is complete; Pour the melted embedding wax into the embedding frame, remove all air bubbles, and then gently place the paraffin-soaked tissue into the embedding frame. Once the paraffin has solidified, the gosling kidney tissue will be embedded in the paraffin. Sectioning: Trim the embedded wax block, place it on a microtome, and cut it into sections. Adjust the thickness to about 5~7μm. Use the tip of a brush to pick up the cut paraffin and place it in water at 45℃ to spread it. Use an adhesive glass slide to absorb the spread wax section and place it in a drying oven at 65℃ for about 5 hours to dry it. Storage: Store the dried slices at room temperature for subsequent experiments.

[0041] 1.3.2.3 HE staining

[0042] Dewaxing: Slices of gosling kidneys were immersed in xylene I and II for 10 minutes each, then in anhydrous ethanol I and II for 10 minutes each, then in 95% and 90% ethanol for 5 minutes each, and finally rinsed with tap water 3 times to remove the ethanol. Hematoxylin staining: Stain gosling kidney sections with hematoxylin for 3 min, then rinse 2-3 times under running water, differentiate with 1% hydrochloric acid alcohol for a few seconds, wash with distilled water for about 30 s, invert blue with 0.5% ammonia water for 10-15 min, and wash with distilled water for 30 s; Eosin staining: Immerse in 70%, 85%, and 95% alcohol for 2 min each, and stain with alcohol and eosin for 45 s. Dehydration: Place in 95% ethanol for 5 min, then in anhydrous ethanol for 5 min; Transparent: Immerse in xylene I and II for 10 min; Sealing: Place a drop of neutral resin on a section of gosling kidney tissue, cover with a coverslip, and let it air dry before storing. Photography: Select a representative field of view from each slide and photograph it using an upright microscope.

[0043] 1.3.2.4 Image Acquisition and Analysis

[0044] Seven kidney tissue samples from goslings were randomly selected from each group for HE staining and image analysis using the Leica Application Suite-LASV4.4 image analysis system.

[0045] 1.2.5 Determination of renal and hepatic function indicators in goslings

[0046] 1.2.5.1 Determination of biochemical indicators of kidney function in goslings

[0047] The biochemical parameters of uric acid (UA), blood urea nitrogen (BUN), and creatinine (CRE) in the kidneys of goslings were tested and calculated according to the instructions and given formulas of each kit.

[0048] 1.2.5.2 Determination of biochemical indicators of liver function in goslings

[0049] The biochemical parameters of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) in gosling livers were tested and calculated according to the instructions and given formulas of each kit.

[0050] 1.2.6 Determination of uric acid metabolism indicators in goslings

[0051] The biochemical indicators of xanthine oxidase (XOD) and adenosine deaminase (ADA) in gosling serum were tested and calculated according to the instructions and formulas of each kit; the biochemical indicators of XOD and ADA in gosling liver were tested and calculated according to the instructions and formulas of each kit.

[0052] 1.2.7 Measurement of Inflammatory Factor-Related Indicators in Goslings

[0053] The levels of inflammatory factors were determined using the ELISA method according to the instructions. The levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the serum of goslings were measured, as well as the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1) in the kidneys of goslings.

[0054] 1.2.8 Determination of antioxidant capacity-related indicators in goslings

[0055] The biochemical parameters of superoxide dismutase (SOD) and malondialdehyde (MDA) in gosling serum were tested and calculated according to the instructions and formulas of each kit; the biochemical parameters of SOD and MDA in gosling kidneys were tested and calculated according to the instructions and formulas of each kit.

[0056] 1.2.9 Determination of related indicators of renal tubular interstitial fibrosis in goslings

[0057] The level of transforming growth factor-β (TGF-β) in the serum of goslings was determined by ELISA according to the instructions.

[0058] 1.2.10 Detection of uric acid metabolism, inflammatory factors, antioxidants, and mRNA expression of genes related to renal tubulointerstitial fibrosis in goslings

[0059] 1) Uric acid production: The expression levels of XOD and ADA gene mRNA in the liver of goslings were measured; Uric acid reabsorption: The expression levels of URAT1 and GLUT9 gene mRNA in the kidney of goslings were measured; Uric acid excretion: The expression levels of OAT1 and OAT3 gene mRNA in the kidney of goslings were measured. 2) Expression of inflammatory factor-related genes: The mRNA expression levels of TLR4, MyD88, NF-κB, NLRP3, ASC, and caspase-1 genes in the kidneys of goslings were measured; 3) Expression of antioxidant-related genes: The mRNA expression levels of Nrf2, HO-1, γ-GCS, and NQO1 genes in the kidneys of goslings were measured; 4) Expression of genes related to renal tubular interstitial fibrosis: The expression levels of COL1A1, ACTA, FN1, CTNNB1, and WNT1 mRNA in the kidneys of goslings were measured.

[0060] The above gene primer designs are shown in Table 4. The full sequences of each gene in geese and other tissues were searched in GenBank on the NCBI website. The primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0061] Table 4 Primer Design

[0062] (1) Extraction of total RNA from liver (kidney)

[0063] ① Pre-cool the mortar with liquid nitrogen, then quickly transfer the gosling liver stored in the -80℃ refrigerator to the pre-cooled mortar. Use a pestle to quickly grind the liver tissue into a white powder, continuously adding an appropriate amount of liquid nitrogen to maintain a low temperature environment. ② Place the ground white powder into an enzyme-free centrifuge tube containing 1 ml of Trizol placed on an ice box, shake for 15 seconds, let stand at room temperature for 10 minutes, and then centrifuge at 1200 g at 4°C for 10 minutes. ③ Carefully aspirate 800 μl of supernatant (homogeneous lysis buffer) from the enzyme-free centrifuge tube ② into a new enzyme-free centrifuge tube.

[0064] ④ Add 200 μl of chloroform to the above homogenate lysis solution, tighten the cap of the enzyme-free centrifuge tube, and shake up and down for 30 seconds until the mixed solution emulsifies into a milky white color; ⑤ After standing at room temperature for 5 minutes, centrifuge at 1200g for 15 minutes at 4℃; ⑥ Collect approximately 200 μl of the upper layer (clear layer) from the centrifuged test tube into a new enzyme-free centrifuge tube; ⑦ Add 200 μl of isopropanol to the supernatant of the deenzyme centrifuge tube in ⑥, invert the deenzyme centrifuge tube to mix the liquid in the deenzyme centrifuge tube evenly, let it stand on an ice box for 10 min, then centrifuge at 1200g, 4℃ for 10 min. A milky white precipitate can be seen at the bottom of the deenzyme centrifuge tube. ⑧ Carefully pour out the supernatant along the wall of the deenzyme-removed centrifuge tube (do not touch the precipitate). Add 1 ml of 75% ethanol, gently invert the tube to wash the wall until a white precipitate floats to the surface, centrifuge at 8000g and 4°C for 5 minutes, then carefully discard the supernatant, again avoiding touching the precipitate.

[0065] ⑨ Finally, discard the 75% ethanol (by volume), dry at room temperature, and allow the precipitate to stand for 3 minutes. After the precipitate has dried, add 20 μl of RNASE-FREE water to dissolve and mix the precipitate.

[0066] (2) Reverse transcription of total RNA from the liver (kidney)

[0067] Using the total RNA extracted in the above experiment as a template, reverse transcription was performed. The steps are as follows: ① Genomic DNA removal: Prepare a mixture in an enzyme-free centrifuge tube. See Table 5 for specific reagent dosages and methods.

[0068] Table 5. Dosage and Method of Genome Removal Reagents

[0069] Gently mix by pipetting.

[0070] ②Preparation of reverse transcription reaction system

[0071] Add the following mixture directly to the reaction tube in step ①. See Table 6 for specific reagents and methods.

[0072] Table 6. Dosage and Method of Reverse Transcription Reagents

[0073] Gently mix by pipetting.

[0074] (3) Real-time fluorescent PCR

[0075] ① Prepare the following mixture in a qPCR tube. See Table 7 for specific reagents and methods.

[0076] Table 7 Real-time Quantitative PCR Reaction System

[0077] ② Perform the quantitative PCR reaction according to the conditions in Table 8.

[0078] Table 8. Reaction conditions for quantitative real-time PCR

[0079] 1.2.11 Immunohistochemical expression levels of α-SMA and COL1A1 in gosling kidneys

[0080] (1) Dewaxing: Place the paraffin sections in xylene I for 10 min → xylene II for 10 min (to remove excess liquid) → anhydrous ethanol I for 10 min → anhydrous ethanol II for 10 min → 95% ethanol for 5 min → 90% ethanol for 3 min → place in PBS buffer.

[0081] (2) Antigen retrieval: α-SMA and COL1A1 antigen retrieval 3-5 min → PBS wash 3 times, 3 min each time → add appropriate amount of endogenous peroxidase blocker → incubate gently at room temperature for 10 min → PBS wash 3 times, 3 min each time.

[0082] (3) Add working solution of normal goat serum for blocking: 100 μl serum → incubate gently at room temperature for 15 min.

[0083] (4) Add primary antibody: Gently shake off the blocking solution → add 100 μl of primary antibody, incubate overnight at 4°C → wash 3 times with PBS, 3 min each time.

[0084] (5) Add biotin-labeled goat anti-mouse / rabbit IgG: Add 100 μl of secondary antibody → incubate gently at room temperature for 15 min → wash 3 times with PBS, 3 min each time.

[0085] (6) Add horseradish enzyme-labeled streptavidin working solution: Add 100 μl of horseradish enzyme-labeled streptavidin working solution → incubate gently at room temperature for 15 min → wash 3 times with PBS, 3 min each time.

[0086] (7) DAB color development: Add DAB color development solution → incubate gently at room temperature for 15 min → wash 3 times with PBS, 3 min each time.

[0087] (8) Counterstaining: Hematoxylin counterstaining for 1 min → differentiation for a few seconds, then invert blue for 10 min.

[0088] (9) Dehydration: Place the alcohol in the following order of alcohol concentration from high to low: 90% alcohol for 5 min → 95% alcohol for 5 min → 100% alcohol I for 10 min → 100% alcohol II for 10 min.

[0089] (10) Transparency: Place the sliced ​​gosling kidneys into xylene I for about 10 minutes, then into xylene II for about 10 minutes, and let them air dry slightly.

[0090] (11) Mounting: Neutral resin for mounting.

[0091] (12) Microscopic examination: Microscopic examination, image acquisition and analysis, and analysis of immunohistochemical stained sections using an image analysis system.

[0092] 1.3 Data Analysis

[0093] The obtained data were imported into SPSS 21.0 analysis software, and the ANOVA one-way ANOVA method was used for data analysis. All data are expressed as x±s (mean ± standard deviation). The experimental results were considered statistically significant with P<0.05. The significant differences among the four groups were analyzed and compared using SPSS. GraphPad Prism 5.0 software was used for statistical processing, and bar charts were generated based on the statistical analysis of the data.

[0094] 2 Results and Analysis

[0095] 2.1 Clinical observation of goslings

[0096] Compared to the control group, goslings in the model group exhibited trembling, lethargy, and weak gait; increased water intake and the excretion of thin, white feces; and copious amounts of white urine adhering to the feathers around the vent. Goslings in the taurine prevention group showed better overall condition and excreted less white, watery feces compared to the model group. The taurine control group showed no apparent changes compared to the normal control group.

[0097] 2.2 Effects of Taurine on the Kidneys and Liver of Goslings with Hyperuricemic Nephropathy

[0098] 2.2.1 Effects of Taurine on Kidney and Liver Indices in Goslings with Hyperuricemic Nephropathy

[0099] Table 9. Effects of taurine on kidney and liver indices in goslings.

[0100] Note: Different subscript letters in the same column indicate significant differences (P < 0.05), and the same subscript letter in the same column indicate no significant differences (P > 0.05) (the same applies below).

[0101] From Table 9 and Figure 1 As can be seen, compared with the control group goslings, the kidney index of the model group goslings was significantly increased (P < 0.05), while the liver index was increased, but not significantly (P > 0.05). In the 0.1% taurine prevention group goslings, both the kidney and liver indices were increased, but there were no significant differences (P > 0.05). In the 0.1% taurine control group goslings, both the kidney and liver indices were decreased, but there were no significant differences (P > 0.05).

[0102] 2.2.2 Effects of Taurine on the Morphological Changes of Kidney Tissue in Goslings with Hyperuricemic Nephropathy

[0103] like Figure 2 As shown, the glomerular capsule wall structure of goslings in the control group remained unchanged. In the model group, compared to the control group, thickening of the glomerular capsule wall, significant swelling of the renal tubular epithelial cells with indistinct cell boundaries, severe glomerular damage and even blockage, nuclear dissolution, and inflammatory cell exudation were observed. The taurine prevention group also showed glomerular capsule wall thickening and renal tubular epithelial cell swelling, but the swelling was milder than in the model group, with obvious signs of recovery. In the taurine control group, the glomerular capsule wall and cell nuclei of goslings exhibited normal morphology and structure, without swelling. The renal tubules showed normal morphology and size without pathological changes, showing no significant difference compared to the normal control group.

[0104] 2.2.3 Effects of Taurine on Kidney and Liver Function Indicators in Goslings with Hyperuricemic Nephropathy

[0105] The levels of UA, BUN, and CRE in the serum of goslings were measured using UA, BUN, and CRE test kits. The average levels of UA, BUN, and CRE in the serum of goslings in each group were taken, and the standard deviation of each group was calculated. The results are shown in Table 10.

[0106] Table 10 Effects of taurine on the levels of UA, BUN, and CRE in the serum of goslings.

[0107] From Table 10 and Figure 3 It can be seen that, compared with the control group goslings, the levels of UA, BUN, and CRE in the model group goslings were significantly increased (P<0.05); the UA level in the 0.1% taurine prevention group goslings was significantly increased (P<0.05), while the levels of BUN and CRE were increased, but there was no significant difference (P>0.05); the UA level in the 0.1% taurine control group goslings was significantly decreased (P<0.05), while the levels of BUN and CRE were decreased, but there was no significant difference (P>0.05).

[0108] The levels of AST, ALT, and LDH in the serum of goslings were measured using AST, ALT, and LDH test kits. The average levels of AST, ALT, and LDH in the serum of goslings in each group were taken, and the standard deviation of each group was calculated. The results are shown in Table 11.

[0109] Table 11 Effects of taurine on the levels of AST, ALT, and LDH in the serum of goslings

[0110] From Table 11 and Figure 4It can be seen that, compared with the control group goslings, the AST and LDH levels in the model group goslings were significantly increased (P<0.05), and ALT levels were also increased, but there was no significant difference (P>0.05); the AST, ALT, and LDH levels in the 0.1% taurine prevention group goslings were all increased, but there was no significant difference (P>0.05); the AST, ALT, and LDH levels in the 0.1% taurine control group goslings were all decreased, but there was no significant difference (P>0.05).

[0111] 2.3 Effects of Taurine on Uric Acid Metabolism in Goslings with Hyperuricemic Nephropathy

[0112] 2.3.1 Uric acid production in goslings

[0113] (1) XOD and ADA content in gosling serum

[0114] The levels of XOD and ADA in the serum of goslings were measured using XOD and ADA test kits. The average values ​​of the XOD and ADA levels in the serum of goslings in each group were taken, and the standard deviation of each group was calculated. The results are shown in Table 12.

[0115] Table 12 Effects of taurine on XOD and ADA levels in gosling serum

[0116] From Table 12 and Figure 5 It was found that, compared with the control group goslings, the serum XOD and ADA levels of the model group goslings were significantly increased (P<0.05); the serum XOD and ADA levels of the 0.1% taurine treatment group goslings were significantly increased, but there was no significant difference (P>0.05); the liver XOD content of the 0.1% taurine control group goslings was decreased, but there was no significant difference (P>0.05), while the ADA content was increased, showing a significant difference (P<0.05).

[0117] (2) XOD and ADA content in gosling liver

[0118] The levels of XOD and ADA in the liver were measured using XOD and ADA test kits. The average levels of XOD and ADA in the livers of goslings in each group were taken, and the standard deviation of each group was calculated. Table 13 is the result.

[0119] Table 13 Effects of taurine on XOD and ADA in the liver of goslings

[0120] From Table 13 and Figure 6It can be seen that, compared with the control group goslings, the XOD and ADA contents in the liver of the model group goslings were significantly increased (P<0.05); however, the XOD and ADA contents in the liver of the 0.1% taurine prevention group goslings were increased, but there was no significant difference (P>0.05); the XOD and ADA contents in the liver of the 0.1% taurine control group goslings were significantly decreased (P<0.05).

[0121] (3) Relative expression levels of XOD and ADA mRNA in the liver of goslings

[0122] Table 14 Effects of taurine on the relative expression levels of genes related to uric acid production in the liver of goslings

[0123] From Table 14 and Figure 7 It can be seen that, compared with the control group goslings, the expression levels of XOD and ADA mRNA in the liver of goslings in the model group were significantly increased (P<0.05); the expression levels of XOD and ADA mRNA in the liver of goslings in the 0.1% taurine prevention group were increased, but there was no significant difference (P>0.05); the expression levels of XOD and ADA mRNA in the liver of goslings in the 0.1% taurine control group were decreased, but there was no significant difference (P>0.05).

[0124] 2.3.2 Uric acid reabsorption in goslings

[0125] Relative expression levels of URAT1 and GLUT9 mRNA in gosling kidneys

[0126] Table 15 Effects of taurine on the relative expression levels of genes related to uric acid reabsorption in gosling kidneys

[0127] From Table 15 and Figure 8 It can be seen that, compared with the control group goslings, the expression levels of URAT1 and GLUT9 mRNA in the kidneys of goslings in the model group were significantly increased (P<0.05); the expression levels of URAT1 and GLUT9 mRNA in the kidneys of goslings in the 0.1% taurine prevention group were significantly increased (P<0.05); and the expression levels of URAT1 and GLUT9 mRNA in the kidneys of goslings in the 0.1% taurine control group were decreased, with no significant difference (P>0.05).

[0128] 2.3.3 Uric acid excretion in goslings

[0129] Relative expression levels of OAT1 and OAT3 mRNA in gosling liver

[0130] Table 16 Effects of taurine on the relative expression levels of genes related to uric acid excretion in gosling kidneys

[0131] From Table 16 and Figure 9 It can be seen that, compared with the control group goslings, the expression levels of OAT1 and OAT3 mRNA in the kidneys of goslings in the model group were significantly decreased (P < 0.05); the expression level of OAT1 mRNA in the kidneys of goslings in the 0.1% taurine prevention group was significantly decreased (P < 0.05), and the expression level of OAT3 mRNA was decreased, but the difference was not significant (P > 0.05), although the expression level was higher than that in the kidneys of goslings in the model group; the expression level of OAT1 mRNA in the kidneys of goslings in the 0.1% taurine control group was decreased, but the difference was not significant (P > 0.05); the expression level of OAT3 mRNA in the kidneys of goslings in the 0.1% taurine control group was increased, but the difference was not significant (P > 0.05).

[0132] 2.4 Effects of Taurine on Inflammatory Pathways in Goslings with Hyperuricemic Nephropathy

[0133] 2.4.1 Serum levels of IL-6, TNF-α, and IL-1β in goslings

[0134] The levels of IL-6, TNF-α, and IL-1β in the serum of goslings were measured using an ELISA kit. The average levels of IL-6, TNF-α, and IL-1β in the serum of goslings from each group were taken, and the standard deviation of each group was calculated. The results are shown in Table 17.

[0135] Table 17 Effects of taurine on serum levels of IL-6, TNF-α, and IL-1β in goslings

[0136] From Table 17 and Figure 10 It can be seen that, compared with the control group goslings, the serum levels of IL-6, TNF-α, and IL-1β in the model group goslings were significantly increased (P < 0.05); the serum levels of IL-6, TNF-α, and IL-1β in the 0.1% taurine prevention group goslings were increased, but the difference was not significant (P > 0.05); the serum levels of IL-6, TNF-α, and IL-1β in the 0.1% taurine control group goslings were decreased, but the difference was not significant (P > 0.05).

[0137] 2.4.2 Levels of TNF-α, IL-1β, and MCP-1 in the kidneys of goslings

[0138] The levels of TNF-α, IL-1β, and MCP-1 in the kidneys of goslings were measured using an ELISA kit. The average levels of TNF-α, IL-1β, and MCP-1 in the kidneys of goslings in each group were taken, and the standard deviation of each group was calculated. The results are shown in Table 18.

[0139] Table 18 Effects of taurine on the levels of TNF-α, IL-1β, and MCP-1 in the kidneys of goslings

[0140] From Table 18 and Figure 11 It can be seen that, compared with the control group goslings, the levels of TNF-α, IL-1β, and MCP-1 in the kidneys of the model group goslings were significantly increased (P < 0.05); at the same time, the increase of TNF-α in the kidneys of the 0.1% taurine prevention group was significantly different (P < 0.05), while the increase of IL-1β and MCP-1 was not significantly different (P > 0.05); the decrease of TNF-α in the kidneys of the 0.1% taurine control group was significant (P < 0.05), while the decrease of IL-1β and MCP-1 was not significantly different (P > 0.05).

[0141] 2.4.3 Relative expression levels of NLRP3, ASC, Caspase1, TLR4, MyD88, and NF-κB mRNA in gosling kidneys

[0142] Table 19 Effects of taurine on the relative expression levels of kidney inflammation-related genes in goslings with hyperuricemic nephropathy

[0143] From Table 19 and Figure 12 It can be seen that, compared with the control group goslings, the expression levels of NLRP3, ASC, Caspase1, TLR4, MyD88, and NF-κB mRNA in the kidneys of goslings in the model group were significantly increased (P < 0.05); in the 0.1% taurine prevention group, the expression levels of Caspase1 and MyD88 mRNA in the kidneys of goslings were increased, but the difference was not significant (P > 0.05), while the expression levels of NLRP3, ASC, TLR4, and NF-κB mRNA were increased, and the difference was significant (P < 0.05); in the 0.1% taurine control group, the expression levels of NLRP3, ASC, Caspase1, and NF-κB mRNA in the kidneys of goslings were decreased, but the difference was not significant (P > 0.05), while the expression levels of TLR4 and MyD88 mRNA were increased, and the difference was significant (P < 0.05).

[0144] 3.5 Effects of taurine on antioxidant pathways in goslings with hyperuricemic nephropathy

[0145] 3.5.1 SOD and MDA content in gosling serum

[0146] The levels of SOD and MDA in the serum of goslings were measured using SOD and MDA test kits. The average levels of SOD and MDA in the serum of goslings in each group were taken, and the standard deviation of each group was calculated. Table 20 is obtained.

[0147] Table 20 Effects of taurine on SOD and MDA levels in gosling serum

[0148] From Table 20 and Figure 13 It can be seen that, compared with the control group goslings, the serum MDA content in the model group goslings was significantly increased (P<0.05), but the SOD content was significantly decreased (P<0.05); in the 0.1% taurine prevention group, the serum MDA content was increased, but the difference was not significant (P>0.05), while the SOD content was decreased, and the SOD difference was significant (P<0.05); in the 0.1% taurine control group goslings, the serum SOD content was increased, and the difference was significant (P<0.05); the MDA content was decreased, but the MDA difference was not significant (P>0.05).

[0149] 2.5.2 SOD and MDA content in gosling kidneys

[0150] The levels of SOD and MDA in the kidneys of goslings were measured using SOD and MDA test kits. The average levels of SOD and MDA in the kidneys of goslings in each group were taken, and the standard deviation of each group was calculated. Table 21 is the result.

[0151] Table 21 Effects of taurine on SOD and MDA content in the kidneys of goslings

[0152] From Table 21 and Figure 14 It can be seen that, compared with the control group goslings, the SOD content in the kidneys of the model group goslings was significantly decreased (P<0.05), and the MDA content was significantly increased (P<0.05); the SOD content in the kidneys of the 0.1% taurine prevention group goslings was significantly decreased (P<0.05), and the MDA content was significantly increased (P<0.05); the SOD content in the kidneys of the 0.1% taurine control group goslings was increased, with no significant difference in SOD content (P>0.05), while the MDA content was decreased, with a significant difference in MDA content (P<0.05).

[0153] 2.5.3 Relative expression levels of NFE2L2, NQO1, γ-GCS, and HO-1 mRNA in gosling kidneys

[0154] Table 22 Effects of taurine on the relative expression levels of antioxidant-related genes in the kidneys of goslings

[0155] From Table 22 and Figure 15It can be seen that, compared with the control group goslings, the expression levels of NFE2L2, NQO1, γ-GCS, and HO-1 mRNA in the kidneys of goslings in the model group were significantly reduced (P < 0.05); the expression levels of NQO1, γ-GCS, NFE2L2, and HO-1 mRNA in the kidneys of goslings in the 0.1% taurine prevention group were significantly reduced (P < 0.05); the expression level of NFE2L2 mRNA in the kidneys of goslings in the 0.1% taurine control group was significantly increased (P < 0.05), while the expression levels of NQO1, γ-GCS, and HO-1 mRNA were not significantly increased (P > 0.05).

[0156] 2.6 Effects of Taurine on the Renal Tubulointerstitial Fibrosis Pathway in Hyperuricemic Goslings

[0157] 2.6.1 TGF-β levels in gosling serum

[0158] The TGF-β level in gosling serum was measured using an ELISA kit. The average TGF-β level in the serum of each group was taken, and the standard deviation of each group was calculated. The results are shown in Table 23.

[0159] Table 23 Effect of taurine on TGF-β content in gosling serum

[0160] From Table 23 and Figure 16 It can be seen that, compared with the control group goslings, the serum TGF-β content in the model group goslings was significantly increased (P<0.05); the serum TGF-β in the 0.1% taurine prevention group goslings was increased, with a significant difference (P<0.05); the serum TGF-β in the 0.1% taurine control group goslings was decreased, with no significant difference (P>0.05).

[0161] 2.6.2 Relative expression levels of COL1A1, ACTA, FN1, CTNNB1, and WNT1 mRNA in gosling kidneys

[0162] Table 24 Effects of taurine on the relative expression levels of genes related to renal tubulointerstitial fibrosis in goslings

[0163] From Table 24 and Figure 17It can be seen that, compared with the control group goslings, the expression levels of COL1A1, ACTA, FN1, CTNNB1, and WNT1 mRNA in the kidneys of goslings in the model group were significantly increased (P < 0.05); in the 0.1% taurine prevention group, the expression levels of ACTA and CTNNB1 mRNA in the kidneys of goslings were increased, but the difference was not significant (P > 0.05), while the expression levels of COL1A1, FN1, and WNT1 mRNA were increased, and the difference was significant (P < 0.05); in the 0.1% taurine control group, the expression levels of COL1A1, FN1, CTNNB1, and WNT1 mRNA in the kidneys of goslings were decreased, but the difference was not significant (P > 0.05), while the expression level of ACTA mRNA was increased, and the difference was significant (P < 0.05).

[0164] 2.6.3 Effects of relative protein expression of COL1A1 and α-SMA in gosling kidneys

[0165] From Table 25 and Figures 18-20 It can be seen that the immunohistochemical results of gosling kidneys show that COL1A1 and α-SMA proteins are expressed to varying degrees in the distal and proximal tubular epithelial cell membranes and cytoplasm of gosling kidney tissues in all groups, and the immunoreaction products of the two proteins are brownish-yellow. The expression intensity of COL1A1 and α-SMA proteins in the kidney tissues of goslings in the normal control group is slightly weaker. Figure 19 C, 20C); COL1A1 and α-SMA proteins showed strong positive expression in the kidney tissue of goslings in the model group (C, 20C); Figure 19 M, 20M). The average optical density values ​​of COL1A1 and α-SMA proteins expressed in the kidney tissues of each group are detailed in Table 25.

[0166] Table 25. Effects of taurine on the expression of COL1A1 and α-SMA, marker proteins of renal tubulointerstitial fibrosis in goslings.

[0167] From Table 25 and Figure 18 The results showed that, compared with the control group goslings, the expression levels of COL1A1 and α-SMA in the kidneys of goslings in the model group were significantly increased (P < 0.05); the expression levels of COL1A1 and α-SMA in the kidneys of goslings in the 0.1% taurine prevention group were significantly increased (P < 0.05); and the expression levels of COL1A1 and α-SMA in the kidneys of goslings in the 0.1% taurine control group were decreased, but the difference was not significant (P > 0.05).

[0168] Example 2

[0169] 1. Main Instruments and Reagents

[0170] 1.1 Main Instruments

[0171] Table 26 Main Instruments Used in the Experiment

[0172] 1.2 Main Reagents

[0173] 1.2.1 Main Reagent Information

[0174] Table 27 Major Reagents and Their Manufacturers

[0175] 1.2.2 Preparation of reagents

[0176] (1) Western Washing Solution (TBST): Pour TBS powder into a clean beaker, add 900ml of double-distilled water and stir until dissolved. Then add 1.5ml of Tween 20 solution. After stirring the solution evenly, bring the volume to 1L with double-distilled water.

[0177] (2) Western electrophoresis buffer: Take one bottle of SDS-PAGE electrophoresis buffer powder, pour it into a clean beaker, add double-distilled water to dissolve it to 1L, which is 5× electrophoresis buffer. Dilute the 5× electrophoresis buffer to 1× electrophoresis buffer, mix well and it is ready for use.

[0178] (3) Transfer solution: 3.03g tris, 14.4g glycine, 200ml anhydrous methanol, and ddH2O to a final volume of 1L.

[0179] 2. WB Experiment Content

[0180] 2.1 Experimental Procedure

[0181] Protein extraction—protein quantification—SDS-PAGE—transfer to PVDF membrane—blocking—incubation with primary antibody—incubation with secondary antibody—ECL substrate luminescence—image saving.

[0182] 2.2 Experimental Procedure

[0183] 2.2.1 Protein Extraction

[0184] (1) Melt the lysis buffer at room temperature in advance, estimate the volume to be used in the experiment, dispense it, and mix in 1% PMSF of the dispensed volume for later use.

[0185] (2) Add the corresponding volume of lysed sample according to the mass and volume of each sample, and then continue to place it on ice for 5 minutes.

[0186] (3) Turn on the low-temperature refrigerated centrifuge, centrifuge at 12000 rpm and 4℃ for 10 min, and separate the supernatant as the obtained protein extract.

[0187] 2.2.2 Protein Quantification

[0188] (1) Preparation of standard curve: Dispense 0.5 μg / μl BSA protein standard solution into each well of the microplate in volumes of 0, 1, 2, 4, 8, 12, 16, and 20 μl, and replenish the remaining volume to 20 μl with PBS buffer.

[0189] (2) Preparation of protein test solution: Mix 1 μl of protein extract from the test sample with 19 μl of PBS buffer.

[0190] (3) BCA reaction: According to the volume ratio of A liquid to B liquid of 50:1, estimate the volume of BCA working solution required for this experiment at 200μl per well. Add the prepared working solution to each well, repeatedly blow and mix with a pipette, and then place it at 37℃ for 20min. At this time, the solution changes from green to purple.

[0191] (4) Data reading: Start the microplate reader 15 minutes in advance to preheat, place the microplate on the stage, set the reading wavelength to 570nm, and record the data.

[0192] (5) Plot a standard curve with standard protein concentration and corresponding absorbance value, calculate the sample protein concentration by regression equation, and multiply by the dilution factor to get the sample protein concentration.

[0193] 2.2.3 SDS-PAGE

[0194] (1) Assemble the electrophoresis apparatus: After cleaning the glass plates, use filter paper to absorb the surface moisture and let them air dry at room temperature. Once you confirm that there are no water stains or dust on the surface, you can begin the assembly. Place the long plate on the outside and the short plate on the inside, align the bottom edges, and fix them with wedges. Seal the bottom edges and clamp both sides together before starting the glue pouring.

[0195] (2) Preparation of polyacrylamide gel: The appropriate concentration of polyacrylamide gel was selected based on the molecular weight of the target protein. In this experiment, the concentration of the stacking gel was 4% and 5%, and the concentration of the separating gel was 6% and 10%. The gel pouring sequence was from bottom to top. First, the separating gel was prepared. Before pouring, APS and TEMED were added, mixed well, and poured along one side of the glass plate. The top layer was sealed with water to promote gel polymerization. After the separating gel polymerized, the water layer was poured off, and the prepared stacking gel was poured in. Before pouring, APS and TEMED were added again, and finally, the opening was sealed with a comb. After waiting for about 30 minutes, the comb was removed, and sample loading began. The formulations of the various polyacrylamide concentrations are as follows: Table 28 Polyacrylamide gel formulation

[0196] Note: Use separating gel buffer when preparing separating gel; use stacking gel buffer when preparing stacking gel; (3) Preparation of protein loading solution: Dilute the protein sample with 5× Loading Buffer and PBS, boil in a boiling water bath for 5 min to prepare the loading solution for later use. The loading volume for this experiment was 20 μl, containing 40 μg of protein.

[0197] (4) SDS-PAGE: After removing the comb, inject the electrophoresis solution into the positive and negative electrodes of the electrophoresis tank respectively, add 20 μl of electrophoresis loading solution to each well, and finally add 5 μl of protein marker. Connect the electrodes, adjust the current to the maximum, the voltage to 80 V, and perform constant voltage electrophoresis for 2.5 h.

[0198] 2.2.4 Transfer

[0199] (1) Preparation: First, after the transfer buffer is prepared, it should be pre-cooled in a 4°C refrigerator; second, when the electrophoresis is about to end, the filter paper, sponge and other materials used for transfer should be fully soaked in the transfer buffer; finally, cut an appropriate size PVDF membrane, mark the front corner of the membrane, wet it with anhydrous methanol, and then immerse it in the transfer buffer and shake it on a shaker.

[0200] (2) Preparation of the “sandwich”: After electrophoresis, remove the glass plate, cut it open with a blade, cut off the bottom of the stacking gel and the separating gel, and peel off the remaining whole piece of gel into a container containing transfer buffer and soak for 10 minutes; first lay a layer of sponge on the negative electrode side of the transfer clamp, then lay 5 layers of filter paper, then lay the gel, followed by the PVDF membrane, at which point the front side of the membrane should be in contact with the gel, and finally lay 5 layers of filter paper and a layer of sponge on top of the PVDF; forming a “sandwich” shape of “sponge-filter paper-membrane-gel-filter paper-sponge”.

[0201] (3) After the “sandwich” is laid out, clamp the transfer clip, insert it into the transfer tank, inject sufficient transfer buffer, turn on the electrode, adjust the current to the maximum, and transfer for 1.5 hours at 80V.

[0202] 2.2.5 Closed

[0203] (1) Preparation: Prepare 5% (M / V) skim milk powder using TBST buffer solution.

[0204] (2) After the transfer is completed, take out the PVDF film, immerse it in TBST, and shake it on a shaker for 5 minutes.

[0205] (3) Discard the TBST, immerse the PVDF membrane in the skim milk powder solution, and shake it slowly on a shaker for 1 hour.

[0206] 2.2.6 Incubation of primary antibody

[0207] (1) Dilute the antibody with 5% (M / V) skim milk powder to prepare antibody working solution.

[0208] (2) Pour the antibody working solution into the hybridization bag, place the sealed PVDF membrane in it, seal it with a film press, and incubate the antibody at 4°C overnight.

[0209] 2.2.7 Incubation of secondary antibodies

[0210] (1) After incubating the primary antibody, the PVDF membrane is taken out of the hybridization bag, immersed in TBST, shaken on a shaker for 5 minutes, and this step is repeated 4 times.

[0211] (2) Dilute the antibody with 5% (M / V) skim milk powder to prepare the secondary antibody working solution.

[0212] (3) Pour the secondary antibody working solution into the hybridization bag, put in the rinsed PVDF membrane, seal it with a film press, and incubate the antibody at 37°C for 45 minutes.

[0213] 2.2.8 ECL substrate luminescence

[0214] (1) After incubating the secondary antibody, the PVDF membrane is taken out of the hybridization bag, immersed in TBST, shaken on a shaker for 5 minutes, and this step is repeated 6 times.

[0215] (2) Mix equal volumes of ECL chemiluminescent reagents A and B for later use.

[0216] (3) Lay a plastic wrap flat on the table, use filter paper to absorb the moisture on the back of the PVDF, then lay it flat on the plastic wrap, sprinkle ECL luminescent liquid evenly, and let it stand for 5 minutes to react.

[0217] (4) Cover it with another plastic wrap, use a glass rod to remove the excess liquid, then transfer it into a dark box and expose it in a darkroom.

[0218] 2.2.9 Antibody stripping

[0219] (1) After exposure, the PVDF film is removed from the plastic wrap, immersed in distilled water, shaken on a shaker for 5 minutes, and repeated twice.

[0220] (2) Pour out the distilled water from the container, add an appropriate amount of stripping solution to completely cover the PVDF membrane, and shake on a shaker for 15 minutes.

[0221] (3) Pour out the stripping solution from the container, rinse briefly with distilled water, replace with fresh distilled water, shake on a shaker for 10 minutes, and repeat twice.

[0222] 2.2.10 Closed

[0223] (1) Preparation: Prepare 5% (M / V) skim milk powder using TBST buffer solution.

[0224] (2) Take out the PVDF membrane, immerse it in TBST, and shake it on a shaker for 5 minutes.

[0225] (3) Discard the TBST, immerse the PVDF membrane in the skim milk powder solution, and shake it slowly on a shaker for 1 hour.

[0226] 2.2.11 Incubate the internal control antibody

[0227] (1) Dilute the antibody with 5% (M / V) skim milk powder to prepare antibody working solution.

[0228] (2) Pour the antibody working solution into the hybridization bag, place the sealed PVDF membrane in it, seal it with a film press, and incubate the antibody at 4°C overnight.

[0229] 2.2.12 Incubation of secondary antibodies

[0230] (1) After incubating the internal control, the PVDF membrane is taken out of the hybridization bag, immersed in TBST, shaken on a shaker for 5 minutes, and this step is repeated 4 times.

[0231] (2) Dilute the antibody with 5% (M / V) skim milk powder to prepare the secondary antibody working solution.

[0232] (3) Pour the secondary antibody working solution into the hybridization bag, put in the rinsed PVDF membrane, seal it with a film press, and incubate the antibody at 37°C for 45 minutes.

[0233] 2.2.13 ECL substrate luminescence

[0234] (1) After incubating the secondary antibody, the PVDF membrane is taken out of the hybridization bag, immersed in TBST, shaken on a shaker for 5 minutes, and this step is repeated 6 times.

[0235] (2) Mix equal volumes of ECL chemiluminescent reagents A and B for later use.

[0236] (3) Lay a plastic wrap flat on the table, use filter paper to absorb the moisture on the back of the PVDF, then lay it flat on the plastic wrap, sprinkle ECL luminescent liquid evenly, and let it stand for 5 minutes to react.

[0237] (4) Cover it with another plastic wrap, use a glass rod to remove the excess liquid, then transfer it into a dark box and expose it in a darkroom.

[0238] 2.2.14 Results Analysis

[0239] The film was scanned, and the optical density values ​​of the target bands were analyzed using a gel imaging system (Gel-Pro-Analyzer software).

[0240] The results are as follows Figure 21As shown in the figure, T1 and T2 represent taurine concentrations of 0.1% and 0.15%, respectively. The effects of 0.1% and 0.15% taurine on EGFR phosphorylation levels in HN goslings were analyzed using Western blotting, yielding protein bands. Gray-scale analysis of the target protein showed that EGFR phosphorylation levels were significantly increased in HN goslings (p < 0.01), and the addition of both 0.1% and 0.15% taurine concentrations restored it to normal levels (P > 0.05).

[0241] The above experiments measured liver and kidney indices, liver and kidney function indicators, and observed morphological changes in the kidneys of goslings. ALT, AST, and LDH are biochemical indicators reflecting liver function; these three enzymes are key enzymes catalyzing the hydrolysis of elemental phosphate. Purine nucleotides are mainly synthesized in the liver and thymus and metabolized in the liver, small intestine, and kidneys; therefore, hyperuricemia (HUA) may cause liver damage. Damaged hepatocytes increase the ability to synthesize ALT, AST, and LDH. In this experiment, the liver index of goslings in the model group was not significantly different from that in the control group (P>0.05), indicating no statistical significance; serum AST and LDH levels in goslings in the model group were significantly elevated. However, the ALT content among the groups showed differences, but these were not statistically significant.

[0242] As hepatitis Nervous (HN) develops, monosodium urate (MSU) deposits in the renal tubulointerstitium, causing inflammation and fibrosis. MSU also contributes to obstructive kidney disease by participating in the formation of uric acid (UA) stones, leading to changes in kidney volume, mass, and structure. This experiment, using renal organ indices, showed that the model group goslings had significantly increased renal indices, indicating potential kidney disease. In contrast, the taurine prevention group and the taurine control group had relatively normal organ indices. This suggests that the taurine prevention group had a certain effect on improving renal indices, indicating that tau may promote UA excretion in the kidneys, thereby slowing MUS deposition and delaying structural changes. UA, BUN, and CRE levels are important indicators of kidney function. The model group goslings showed significantly elevated serum UA, BUN, and CRE levels, suggesting that the high-calcium, high-protein diet caused some damage to the kidneys, possibly due to urate deposition. The taurine prevention group showed lower serum UA, BUN, and CRE levels compared to the model group, indicating that tau has a certain protective effect on the kidneys. In addition, this experiment also investigated the effects of tau on the kidney pathology of goslings using HE staining. The results showed that, compared to the control group, the model group exhibited severe tubular dilation, a reduced number of glomeruli (some even dissolved), and extensive inflammatory cell infiltration in the interstitium under an upright microscope, indicating that feeding high-calcium, high-protein diets damaged the kidneys of goslings. Meanwhile, the degree of kidney pathological damage in the taurine prevention group was between that of the control and model groups. The taurine prevention group reduced glomerular capsule wall thickening, alleviated tubular dilation, showed a lower number and less structural damage to glomeruli, fewer inflammatory cells, and a relatively clear and intact kidney tissue structure. This suggests that tau has a certain preventive effect on kidney damage in goslings with hepatitis Nervous (HN). Furthermore, the morphological changes in each group were consistent with the aforementioned blood biochemical indicators. This preliminarily indicates that tau has a good preventive effect on HN in goslings, reversing kidney damage to some extent, reducing UA levels in the body, thereby slowing the accumulation of UA and mitigating further damage to the kidneys caused by HUA.

[0243] In this experiment, the results of serum XOD and ADA levels, liver XOD and ADA activity assays, and gene expression detection in goslings showed that compared with the control group, the levels of XOD and ADA in the liver and the expression levels of XOD and ADA mRNA in the model group were significantly increased (P < 0.05). In the taurine prevention group, the levels of XOD and ADA in serum and liver were significantly lower than those in the model group, and the expression levels of XOD and ADA mRNA were also significantly lower (P < 0.05), indicating that tau inhibited the activity of XOD and ADA, thereby reducing UA production in the body. Meanwhile, there was no significant difference in XOD and ADA levels in serum and mRNA expression between the taurine control group and the control group, but the activity of XOD and ADA in the liver was significantly reduced. This further demonstrates that tau can control UA levels and XOD and ADA activity in the liver of goslings through the UA synthesis pathway, effectively reducing UA activity. Therefore, tau can reduce UA production by decreasing the activity of XOD and ADA in the liver.

[0244] This study investigated the mechanism of uric acid transporter proteins in the kidneys of goslings. Compared to the control group, the expression levels of URAT1 and GLUT9 mRNA in the kidneys of goslings in the model group were significantly increased (P < 0.05), while the taurine prevention group decreased the expression of UA reabsorption proteins URAT1 and GLUT9 mRNA in the kidneys of goslings. Compared to the control group, the expression levels of OAT1 and OAT3 mRNA in the model group were significantly decreased (P < 0.05), while the taurine prevention group increased the expression of uric acid excretion proteins OAT1 and OAT3 mRNA in the kidneys of HN goslings. Meanwhile, the UA transporter mRNA expression level in the taurine control group was lower than that in the control group. Therefore, this study demonstrates that tau exerts its UA-lowering effect by acting on UA ​​synthesis, reabsorption, and excretion pathways.

[0245] This experiment showed that, compared with the control group, the model group goslings had significantly higher levels of IL-6, TNF-α, and IL-1β in serum and TNF-α, IL-1β, and MCP-1 in kidneys, as well as significantly higher levels of NLRP3, ASC, Caspase-1, TLR4, MyD88, and NF-κB mRNA in kidneys (P < 0.05) by ELISA and qRT-PCR. In contrast, the taurine prevention group showed significantly lower levels of these levels compared to the model group (P < 0.05). This indicates that MSU crystals activated the NLRP3 inflammasome, and the taurine prevention group significantly reduced NLRP3 expression, suggesting that taurine may inhibit the activation of the NLRP3 inflammasome, thereby inhibiting IL-1β production and reducing inflammatory responses. The taurine control group showed a decrease compared to the normal control group (P > 0.05), indicating that 0.1% taurine has no side effects on the body and can reduce the level of inflammation. In summary, this indicates that taurine participates in the regulation of inflammatory factors and prevents the pathological process of hemangioma induced by high-calcium and high-protein diets in goslings.

[0246] The results of this experiment showed that, compared with the control group, the expression levels of Nrf2, NQO1, γ-GCS, and HO-1 mRNA in the kidney tissue of goslings in the model group were significantly decreased (P < 0.05), the SOD content in the serum and kidney of goslings was significantly decreased (P < 0.05), and the MDA content in the serum and kidney of goslings was significantly increased (P < 0.05). In contrast, the taurine prevention group showed higher expression levels of Nrf2, NQO1, γ-GCS, and HO-1 mRNA in the kidney, and higher SOD content in the serum and kidney of goslings than the model group, while lower MDA content in the serum and kidney of goslings in the model group. These results indicate that taurine enhances the antioxidant capacity of serum and kidney in HN goslings, and the mechanism of taurine's kidney protection is related to its antioxidant effect.

[0247] In this study, we found that compared with the control group goslings, the mRNA expression levels of COL1A1, ACTA, FN, CTNNB1, and WNT1 in the kidneys of the model group goslings were significantly increased (P < 0.05). ELISA results showed that the serum TGF-β level in the model group goslings was significantly increased compared with the control group goslings (P < 0.05). Numerous brownish-yellow granules were observed in the renal tubules and interstitium of the model group goslings' kidney tissue, indicating enhanced expression of COL1A1 and α-SMA proteins in the kidney tissue (P < 0.05). However, these levels decreased in the taurine prevention group compared to the model group (P < 0.05), indicating that taurine can inhibit the expression of COL1A1 and α-SMA proteins and suppress the Wnt / β-catenin signaling pathway. There were no significant changes in the results between the control group goslings and the taurine control group goslings, indicating that 0.1% taurine has no adverse effects on the body. Therefore, taurine regulates the occurrence and development of RIF by inhibiting the Wnt / β-catenin pathway.

[0248] In summary, 1. Taurine can significantly reduce the kidney index in HN goslings, significantly alleviate the degree of morphological lesions, and improve the kidney and liver function levels in HN goslings. This indicates that taurine has a protective effect on the kidneys and liver of HN goslings, reducing kidney and liver function damage in HN model goslings.

[0249] 2. Taurine can reduce UA production and promote UA excretion in HN goslings, indicating that taurine can reduce serum UA levels in HN model goslings.

[0250] 3. Taurine can downregulate the TLR4 / MyD88 / NF-κB and NLRP3 / ASC / Caspase-1 inflammation-related pathways in the kidney tissue of HN goslings, enhance the Keap-1 / Nrf2 / HO-1 antioxidant damage pathway, and delay the Wnt / β-catenin renal tubular interstitial fibrosis pathway, indicating that tau can reduce the inflammatory response in HN goslings, enhance antioxidant damage capacity, and inhibit the process of renal tubular interstitial fibrosis.

[0251] Therefore, taurine can reduce UA production and promote UA excretion, thereby protecting the kidneys of HN goslings by alleviating inflammatory responses, resisting oxidative damage, and delaying the process of renal tubular interstitial fibrosis.

[0252] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of taurine in the preparation of drugs for preventing gosling hyperuricemia nephropathy through target EGFR.

2. Use according to claim 1, wherein The taurine is added to the gosling daily age.

3. Use according to claim 2, wherein the compound is ###0002### The added amount of the taurine in the gosling daily ration is 0.05%-0.15%.

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