Use of an agent that inhibits expression of il4il in the manufacture of a medicament for treating renal fibrosis

Abstract

The application discloses application of a preparation for inhibiting IL4I1 expression in preparation of a medicine for treating renal fibrosis, and belongs to the technical field of biological medicine.The application finds through in-vivo and in-vitro experiments that IL4I1 is closely related to kidney injury and renal fibrosis, and is a potential target for treating kidney injury and renal fibrosis.IL4I1 inhibitors are substances capable of inhibiting IL4I1 enzyme activity, and the in-vivo and in-vitro experimental results show that, for example, extract of plantago, salted plantago extract, verbascoside, isoverbascoside and akebone phenylethanoid glycoside B are all beneficial to relieving renal fibrosis.The application provides theoretical support for developing a medicine for treating renal function impairment and treating and / or relieving renal fibrosis disease by inhibiting IL4I1.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of agents that inhibit IL4I1 expression in the preparation of drugs for treating renal fibrosis. Background Technology

[0002] Renal fibrosis is a common pathological feature of chronic kidney disease (CKD) progressing to end-stage renal disease. Intervention and treatment are crucial for halting and slowing the progression of CKD, but specific treatments are currently lacking. Tryptophan metabolism plays a vital role in renal fibrosis. Tryptophan, through host or microbial metabolism, can generate a series of tryptophan metabolites, which act as ligands to activate the acute renal fibrosis response (AHR), leading to upregulation at the protein and transcriptional gene levels. Simultaneously, it promotes the AHR's entry into the cell nucleus, subsequently triggering the release of downstream signaling molecules and exerting a pro-fibrotic effect.

[0003] Interleukin-4-induced-1 (IL4I1) is a newly discovered tryptophan-metabolizing enzyme that breaks down tryptophan into metabolites such as indole-3-lactic acid and indole-3-propionic acid in tumor cells. Currently, most reports on IL4I1 focus on its roles in tumors and immune regulation; however, its role in renal fibrosis and whether intervention with IL4I1 can alleviate renal fibrosis have not been reported. Summary of the Invention

[0004] The purpose of this invention is to provide the application of agents that inhibit IL4I1 expression in the preparation of drugs for treating renal fibrosis, thereby addressing the problems existing in the prior art. In vitro and in vivo experiments have shown that IL4I1 is closely related to kidney injury and renal fibrosis, and is a potential target for the treatment of kidney injury and renal fibrosis. This invention provides theoretical support for the development of drugs that treat renal impairment, and treat and / or alleviate renal fibrosis by inhibiting IL4I1.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the use of an agent that inhibits IL4I1 expression in the preparation of a drug for treating renal fibrosis.

[0007] This invention also provides the application of IL4I1 as a target in screening drugs for the treatment of renal fibrosis.

[0008] The present invention also provides a method for screening candidate drugs for treating renal fibrosis, comprising applying the drug to be screened to a cell model or animal model of renal fibrosis, detecting the expression level of IL4I1 in the cell model or animal model, and if the expression level of IL4I1 decreases, then the drug to be screened is a candidate drug for treating renal fibrosis.

[0009] Furthermore, the cell model is a kidney cell model.

[0010] Furthermore, the kidney cells include NRK-52E cells.

[0011] The present invention also provides the application of reagents for detecting IL4I1 expression in the preparation of a test kit for assessing the effectiveness of drug treatment for renal fibrosis.

[0012] The present invention discloses the following technical effects:

[0013] This invention, through in vitro and in vivo experiments, has revealed a close association between IL4I1 and kidney injury and renal fibrosis, making it a potential therapeutic target for these conditions. IL4I1 inhibitors are a class of substances that can suppress IL4I1 expression. In vitro and in vivo experimental results show that extracts such as Plantago asiatica extract, salt-processed Plantago asiatica extract, verbascoside, isoverascoside, and akebia phenylethanol glycoside B are all beneficial in alleviating renal fibrosis.

[0014] This invention provides theoretical support for the development of drugs that treat renal impairment by inhibiting IL4I1, and for treating and / or alleviating renal fibrosis. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The protein expression levels of IL4I1 and FN in kidney samples from clinical CKD patients are shown in Figure 1. A represents the results of double immunofluorescence staining of IL4I1 and FN; B represents the relative expression level of IL4I1; C represents the relative expression level of FN. Compared with the normal group, * represents P<0.05; ** represents P<0.01; *** represents P<0.001.

[0017] Figure 2 The values ​​represent the kidney index (A), blood urea nitrogen (B), and serum creatinine biochemical indicators (C) in rats with renal fibrosis; compared with the normal group, * represents P<0.05; ** represents P<0.01; *** represents P<0.001;

[0018] Figure 3 Staining of pathological sections of kidneys from rats with renal fibrosis;

[0019] Figure 4The values ​​represent the IL4I1 protein levels in serum, kidney, and NRK-52E cells of rats with renal fibrosis; where A represents the IL4I1 level in serum; B represents the IL4I1 level in kidney; and C represents the IL4I1 level in NRK-52E cells. Compared with the normal group, * represents P<0.05; ** represents P<0.01; and *** represents P<0.001.

[0020] Figure 5 This study investigated the expression of IL4I1 protein in the kidneys of rats with a renal fibrosis model. In the table, A shows the IL4I1 protein expression bands detected by Western blotting after administration of *Plantago asiatica* and *Plantago saponin*; B shows the relative quantitative analysis of IL4I1 protein expression; and C shows the relative quantitative analysis of IL4I1 gene expression detected by qPCR. In B and C, RH represents the *Plantago asiatica* group, and PH represents the *Plantago saponin* group. Compared with the control group, # represents P < 0.05; ## represents P < 0.01; ### represents P < 0.001; compared with the model group, * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001.

[0021] Figure 6 The effect of *Plantago asiatica* and *Plantago saponin* administration on IL4I1 expression in NRK-52E cells was investigated. A shows the IL4I1 protein expression bands in NRK-52E cells detected by Western blotting; B shows the relative quantitative analysis of IL4I1 protein expression in NRK-52E cells using Western blotting. In B, RH represents the *Plantago asiatica* group, and PH represents the *Plantago saponin* group. Compared with the control group, # represents P < 0.05; ## represents P < 0.01; ### represents P < 0.001; compared with the model group, * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001.

[0022] Figure 7 The expression of IL4I1 in NRK-52E cells after inducing renal fibrosis was measured. In this study, A shows the IL4I1 protein expression band in NRK-52E cells detected by Western blotting; B shows the relative quantitative analysis of IL4I1 protein expression in NRK-52E cells using Western blotting. Compared with the control group, # represents P < 0.05; ## represents P < 0.01; ### represents P < 0.001; compared with the model group, * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001.

[0023] Figure 8The expression of renal fibrosis-related proteins after IL4I1 knockout in NRK-52E cells was analyzed. In this study, A shows the IL4I1 protein expression band in NRK-52E cells detected by Western blotting; B shows the relative quantitative analysis of IL4I1 protein expression in NRK-52E cells using Western blotting; C shows the relative quantitative analysis of FN protein expression in NRK-52E cells using Western blotting. Compared with the WT group, * represents P<0.05; ** represents P<0.01; *** represents P<0.001.

[0024] Figure 9 The expression of renal fibrosis-related transcription factors after IL4I1 overexpression in NRK-52 cells was analyzed. In the data, A represents the relative quantitative analysis of IL4I1 gene expression detected by qPCR; B represents the relative quantitative analysis of FN gene expression detected by qPCR; C represents the relative quantitative analysis of Collagen I gene expression detected by qPCR; D represents the relative quantitative analysis of Vimentin gene expression detected by qPCR; and E represents the relative quantitative analysis of α-SMA gene expression detected by qPCR. Compared with the WT group, * represents P<0.05; ** represents P<0.01; and *** represents P<0.001. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the specification of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0029] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0030] Examples

[0031] I. Experimental materials

[0032] 1. Experimental animals

[0033] 32 male SPF-grade Sprague Dawley (SD) rats, purchased from Shanghai Slack Experimental Animal Co., Ltd., with a body weight of 160 - 180 g. Production license number: SCXK (Shanghai) 2022 - 0004. The animal experiment was approved by the Animal Ethics Committee of Zhejiang Chinese Medical University, approval number: IACUC - 20220829 - 12. Before the start of the experiment, the animals were housed in an environment of 23 - 25 °C with a 12-hour alternating cycle of darkness / light. During the experiment, sufficient food and water were provided to the animals.

[0034] 2. Drug preparation

[0035] Modeling drug: Adenine powder was dissolved in 0.5% CMC - Na solution, mixed and shaken well to prepare a medicinal liquid with a concentration of 150 mg / mL, and stored in a 4 °C refrigerator.

[0036] Alcohol extracts of raw and salt-roasted Plantago asiatica: Raw Plantago asiatica and salt-roasted Plantago asiatica were powdered and sieved, extracted twice with 70% ethanol by reflux, and the extract was concentrated by reflux and dried to obtain an extract. The yield of the raw product was 4.86%; the yield of the salt-roasted product was 7.1%. The extract was dissolved in 0.5% CMC - Na solution, and according to the extraction yield of Plantago asiatica, it was converted to prepare a medicinal liquid with a raw drug amount of 10 g / mL.

[0037] 3. Clinical samples

[0038] Renal tissue biopsy samples were from the Nephrology Department of Zhejiang Provincial Hospital of Traditional Chinese Medicine. The disease group was renal tissue with CKD stages 3 - 5; the normal control group was renal tissue with normal glomerular filtration rate.

[0039] II. Experimental methods

[0040] 1. Establishment of a renal fibrosis animal model and grouped administration

[0041] Male SD rats were randomly divided into four groups: a normal group, a model group, a raw product group, and a salt-processed product group, with eight rats in each group. After one week of acclimatization, rats in the model group, raw product group, and salt-processed product group were administered 150 mg / kg adenine by gavage to induce the plant model. Simultaneously, the raw product group was also administered raw Plantago asiatica extract at a dose of 60 g / kg (once daily); the salt-processed product group was administered salt-processed Plantago asiatica extract at a dose of 60 g / kg (once daily). During the 28-day experiment, the rats were weighed and their weight recorded daily, and they were provided with ample normal food and water.

[0042] 2. Serum biochemical index detection

[0043] The levels of creatinine (CREA) and blood urea nitrogen (BUN) in serum were detected using a fully automated biochemical analyzer.

[0044] 3. Kidney tissue staining

[0045] 3.1 Fixation and embedding of sections

[0046] (1) Fix the kidney tissue sample with 10% formalin for one week; (2) Remove the kidney tissue from the fixative, cut a section of kidney tissue with a thickness of about 4 mm, place it in an embedding cassette, and rinse with running tap water for more than 2 hours; (3) 40% ethanol - 20 min; (4) 70% ethanol - 15 min; (5) 80% ethanol - 10 min; (6) 95% ethanol I - 10 min; (7) 95% ethanol II - 10 min; (8) 100% ethanol I - 10 min; (9) 100% ethanol II - 10 min; (10) 100% ethanol III for 10 min; (11) Stearic acid: paraffin (1:1; 60℃) for 30 min; (12) Paraffin (60℃) for 40 min; (13) Embedding: Take out the tissue and place it at the bottom of the iron box. Carefully pour in the hot paraffin solution, being careful not to shake the tissue. Place the embedded paraffin block on the freezing stage and remove the embedding groove after solidification; (14) Sectioning: Before slicing, freeze the paraffin block containing the kidney tissue sample in a -20℃ freezer for more than 40 min. Set the section thickness to 4 μm. Place the tissue section in a water bath at 37℃. After the tissue section is flattened, adhere it to a glass slide and then place it in an oven at 60℃ for 6 h. After the paraffin on the glass slide melts, store it for later use.

[0047] 3.2 HE (Hematoxylin-eosin) staining

[0048] (1) Before staining, place the tissue sections in an oven at 60°C to melt the paraffin for 2 hours; (2) while hot, place them in xylene solution for 20 minutes; (3) 100% ethanol I for 5 minutes; (4) 100% ethanol II for 5 minutes; (5) 95% ethanol for 5 minutes; (6) 90% ethanol for 5 minutes; (7) 85% ethanol for 5 minutes; (8) rinse with tap water under a gentle flow for 3 minutes; (9) incubate with hematoxylin for 10 minutes; (10) rinse with running water for 8 minutes; (11) stain with eosin for 2 minutes; (12) rinse with running water for 5 minutes; (13) 75% ethanol for 10 seconds; (14) 95% ethanol for 10 seconds; (15) 100% ethanol for 10 seconds; (16) xylene for 10 minutes; (17) mount with neutral resin and observe under an optical microscope.

[0049] 3.3 Masson staining

[0050] The sections were dewaxed and stained according to the kit instructions, following the sequence of Weigert iron hematoxylin staining, acid ethanol differentiation, Masson blue solution for blue reversion, Ponceau S and fuchsin staining, washing with weak acid working solution, phosphomolybdic acid washing, aniline blue staining, dehydration, xylene clearing, and mounting with neutral resin. The sections were then observed under an optical microscope.

[0051] 3.4 Immunofluorescence staining

[0052] (1) Dewax the sections and dewax them with xylene and alcohol according to step 3.2; (2) Repair the sections with 1X antigen retrieval solution in a microwave oven and wash with PBS for 5 min × 3 times; (3) Block with 5% goat serum for 1 h and apply primary antibody overnight at room temperature; (4) Wash with PBS for 5 min × 3 times and incubate with secondary antibody at room temperature in the dark for 2 h; (5) Mount the sections with anti-fluorescence quencher (containing DAPI) and observe under a microscope.

[0053] 4. TGF-β stimulation of rat renal tubular epithelial cells NRK-52E to induce renal fibrosis cell model

[0054] NRK-52E cells in logarithmic growth phase were seeded in 6-well plates (density 2 × 10⁻⁶). 5 Cells were divided into 7 groups (cells / well): blank group, model group, raw plantago group, salt plantago group, verbascoside group, isoverascoside group, and akebia phenylethanoid glycoside group (see Table 1). Each group had 3 replicates. After the cells were stably adhered, the medium was replaced with serum-free medium. The model group was induced with TGF-β1 (10 ng / mL) for 24 h, and then cultured in the corresponding drug-containing serum-free medium for another 24 h.

[0055] Table 1 Cell drug administration groups

[0056]

[0057] 5. Western blot detection

[0058] 5.1 Detection of tissue protein expression

[0059] Total protein extraction from tissue: Weigh 20 mg of renal cortex tissue, add 100 μL of lysis buffer (RIPA:PMSF = 100:1, v / v) per 10 mg of tissue, add magnetic beads, and homogenize the tissue using a pre-cooled cell disruptor until no obvious tissue blocks remain. Centrifuge at 15000 rpm for 10 min and collect the supernatant. Add 5× Loading buffer (sample:Loading buffer = 4:1, v / v) to the protein sample, vortex to mix, place in a 100℃ metal bath to denature the protein, and then store in a -80℃ freezer. After determining the protein concentration of the sample using BCA, perform SDS-PAGE electrophoresis, transfer to a membrane, block, incubate with primary antibody, incubate with secondary antibody, develop, and detect the expression of the target protein.

[0060] 5.2 Detection of cellular protein expression

[0061] Total cellular protein extraction: Discard the culture medium, wash three times with pre-chilled PBS along the cell wall, add 1 mL of PBS solution to each well, collect the cell suspension from each group, centrifuge at 5000 rpm for 5 min at 4°C, and discard the supernatant PBS solution. Add 30 μL of cell lysis buffer to the cell pellet and place on ice, vortex once every 10 min, repeat 3 times, then centrifuge at 15000 rpm for 15 min at 4°C, and transfer the supernatant to a new EP tube for later use. Take a small amount to determine the concentration, and aliquot the rest and store at -80°C to avoid repeated freeze-thaw cycles that could cause protein degradation. After determining the BCA protein concentration in the sample, Western blot was used to detect the expression of the target protein.

[0062] 6. ELISA testing

[0063] Rat serum, kidney, and NRK-52E cell samples were processed according to the ELISA instructions, and the protein content of IL4I1 was detected according to the instructions.

[0064] 7. qPCR detection

[0065] Total RNA was extracted from cells according to the kit instructions. After determining the RNA concentration, reverse transcription and amplification were performed according to the kit instructions. GAPDH was used as an internal control to determine the relative expression level of mRNA.

[0066] 8. CRSPR / Cas9 knockdown of IL4I1

[0067] Primer design for sgIL4I1 was performed, and the primer sequence (sgIL4I1: gcagcgtccgaccacgtat, SEQ ID NO. 1) was synthesized. PCR amplification, ligation, transformation, and culture were carried out by shaking. The culture was then plated on ampicillin-containing plates and incubated for 12-16 hours. Single colonies were selected and amplified. Plasmids were extracted according to the instructions of the endotoxin-free plasmid extraction kit and sequenced. The plasmid with correct sequencing was the constructed IL4I1 knockout plasmid.

[0068] 293T cell lentivirus packaging: 293T cells were plated in antibiotic-free DMEM medium until 90-100% confluence. The transfection complex was prepared as follows: 1200 ng VSVG, 2400 ng PSPA, and 4800 ng target plasmid were added to 0.5 mL of serum-free, antibiotic-free Opti-MEM medium. After mixing by pipetting, 18 μL of PEI 40000 infection aid was added, and the mixture was incubated at room temperature for 20 min. The transfection complex was then evenly added to the 293T cells and cultured at 37°C in a 5% CO2 incubator for 8 h. After 8 h, the medium was replaced with standard DMEM complete medium. After 48 h, the supernatant was collected and concentrated overnight with 5X PEG8000 (medium:PEG8000 ratio 4:1). Centrifuge at 4800 rpm for 10 min at 4℃, discard the supernatant, resuspend the virus in 400 μL PBS, aliquot, and store at -80℃.

[0069] Lentiviral infection of NRK-52E cells: NRK-52E cells were seeded in 24-well plates using DMEM medium without antibiotics. On the second day, polybrene, an infection enhancer, was added to the medium at a ratio of 1:1000, with 400 μL of medium and 100 μL of lentivirus added to each well. After 8 hours, the medium was replaced with regular DMEM complete medium. Once the cells had reached confluence, they were seeded into 6-well plates or T25 cells and continuously killed with 1 ng / mL puromycin. Successfully transfected cells were retained and subjected to subsequent Western blotting to obtain a stable NRK-52E cell line with IL4I1 knockout.

[0070] 9. IL4I1 overexpression

[0071] The rat-derived IL4I1 cDNA cloning plasmid was ordered from the company. Subsequently, lentivirus was packaged into 293T cells, and the lentivirus was used to infect NRK-52E cells in the same manner as the knockout cell line construction.

[0072] III. Results

[0073] 1. High expression of IL4I1 protein in the kidneys of CKD patients

[0074] Immunofluorescence staining was performed on the kidneys of clinical CKD patients and normal controls. Immunofluorescence double staining of IL4I1 and fibronectin FN was performed, as shown in Table 2. Figure 1 As shown, the expression of fibrosis-associated protein FN in the kidneys of clinical CKD patients was higher than that in normal controls, indicating that CKD patients had reached the end stage of chronic kidney disease; at the same time, the clinical samples showed high expression of IL4I1, indicating that IL4I1 is closely related to renal fibrosis, which may serve as a starting point for the treatment of chronic kidney disease.

[0075] Table 2. Statistics of average fluorescence intensity in immunofluorescence.

[0076]

[0077] 2. General behavior and blood biochemical changes in an adenine-induced rat model of renal fibrosis.

[0078] The normal group of rats exhibited good appetite, agile movement, and normal urination and defecation. After adenine gavage, the rats began to show lethargy, sluggishness, frequent curling up, weight loss, increased thirst, increased urine output, and soft, wet stools that easily led to damp bedding after the second week. At the start of the experiment, there was no significant difference in the initial body weight of the rats in each group. Table 3 and Figure 2 The data shows the rat kidney index, serum urea nitrogen, serum creatinine, and serum IL4I1 levels. Figure 2 As shown, compared with the normal group, the levels of kidney index, creatinine, and blood urea nitrogen in the serum of rats in the model group were significantly increased (***P<0.001).

[0079] Table 3. Rat kidney index, serum blood urea nitrogen (BUN), serum creatinine (CARE), and serum IL4I1 levels.

[0080]

[0081]

[0082] 3. Pathological changes in kidney tissue of rats with renal fibrosis model

[0083] Kidney pathology sections stained with HE as follows Figure 3 As shown, the kidneys of the normal group rats had clear and tightly arranged structures, with no abnormalities observed in the renal tubules, glomeruli, or interstitium. In the model group, the kidney tissue structure was disordered, with vacuolar formation. Abundant brownish-yellow 2,8-dihydroxyadenine deposits were visible in the renal tubules. The number of glomeruli was reduced and they were atrophied. The renal tubules were dilated and irregularly arranged. According to Masson staining results, the normal group had a small amount of fibrous tissue around the glomeruli, while the model group had significant blue-stained collagen deposits around the glomeruli and renal tubules, indicating renal interstitial fibrosis.

[0084] 4. ELISA kit was used to detect the IL4I1 protein content in serum, kidney, and NRK-52E cells of rats with renal fibrosis model.

[0085] After the renal fibrosis model was successfully established, its IL4I1 content is shown in Table 4 and Figure 4 The levels of IL4I1 in the serum and kidneys of rats with renal fibrosis were elevated; the levels of IL4I1 in rat renal tubular cells NRK-52E were also significantly increased after stimulation with TGF-β.

[0086] Table 4. ELISA detection of IL4I1 protein levels in rat serum, kidney, and cells.

[0087]

[0088] 5. Western blot analysis of IL4I1 protein and transcription levels in kidney tissues of rats in the renal fibrosis model group and treatment group.

[0089] The expression levels of IL4I1 protein and transcription in a rat model of adenine-induced renal fibrosis were detected by Western blotting (WB) and qPC. Figure 5 Compared to the control group, the protein and transcriptional levels of IL4I1 in the model group were significantly increased. However, fresh plantain reduced the protein level of IL4I1 in the model, while salt plantain significantly reduced both the protein and transcriptional levels of IL4I1 in the model, indicating that salt plantain was more effective than fresh plantain in reducing IL4I1 in the renal fibrosis model.

[0090] Table 5. Western blot analysis of IL4I1 protein and mRNA levels in the kidneys of rats after administration of Plantago asiatica (n=8)

[0091]

[0092]

[0093] 6. Western blot analysis of IL4I1 protein expression in TGF-β-induced NRK-52E cell renal fibrosis model group and treatment group.

[0094] Renal fibrosis was induced in NRK-52E cells by TGF-β. Administration of raw psyllium husk (Raw PS) and propionibacterium saline (Pro PS) alleviated renal fibrosis. Western blotting was used to detect changes in IL4I1 protein expression levels in the renal fibrosis model after administration of psyllium husk. It was found that IL4I1 protein levels significantly increased after TGF-β-induced renal fibrosis in NRK-52E cells, while administration of psyllium husk reduced IL4I1 protein levels after alleviating renal fibrosis (see Table 6). Figure 6 As shown.

[0095] Table 6 shows the protein expression of IL4I1 in NRK-52E cells after administration of Plantago asiatica by Western blotting.

[0096]

[0097] 7. Western blot analysis of IL4I1 protein expression in TGF-β-induced NRK-52E cell renal fibrosis model group and in the treatment groups of verbascoside, isorabatoside, and akebia phenylethanol glycoside B.

[0098] Western blotting was used to detect changes in IL4I1 protein expression levels in a renal fibrosis model after administration of akebia phenethyl glycoside B (Cal B), isovarenoside (Iso), and verbascoside (Ace). It was found that IL4I1 protein levels were significantly increased after NRK-52E cells were induced to develop renal fibrosis by TGF-β, while administration of akebia phenethyl glycoside B, isovarenoside, and verbascoside alleviated renal fibrosis and reduced IL4I1 protein levels, as shown in Table 7. Figure 7 As shown.

[0099] Table 7. IL4I1 protein expression in NRK-52E cells after administration of phenylethanol glycoside B, isochoria, and citrinum.

[0100]

[0101] 8. Western blot analysis of the expression of renal fibrosis-related proteins in NRK-52E cells after IL4I1 knockout.

[0102] Knockdown of IL4I1 in NRK-52E cells resulted in a decrease in the expression of renal fibrosis-related proteins. Furthermore, after TGF-β stimulation to induce a model, the expression of both renal fibrosis-related proteins and IL4I1 significantly increased in unknockdown cells (WT), while TGF-β stimulation did not induce the expression of fibrosis-related proteins in IL4I1-knockdown cells (KO), as shown in Table 8. Figure 8 This indicates that IL4I1 is closely related to renal fibrosis and can serve as a potential target for the treatment of renal fibrosis.

[0103] Table 8. Expression of fibrosis-related proteins after IL4I1 knockout

[0104]

[0105] 9. qPCR detection of renal fibrosis-related transcription factors in NRK-52 cells after overexpression of IL4I1.

[0106] In NRK-52E cells, overexpression of IL4I1 led to an increase in the transcriptional levels of renal fibrosis-related factors (see Table 9). Figure 9 Furthermore, this indicates that IL4I1 is associated with renal fibrosis.

[0107] Table 9. Expression of fibrosis-related proteins after IL4I1 overexpression.

[0108]

[0109] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method of screening for a candidate drug for treating renal fibrosis, characterized by, The method comprises the following steps: applying a drug to be screened to a cell model or an animal model of renal fibrosis; detecting the expression amount of IL4I1 in the cell model or the animal model; and determining that the drug to be screened is a candidate drug for treating renal fibrosis if the expression amount of IL4I1 is reduced. The cell model is a kidney cell model.

2. The method of claim 1, wherein, The kidney cell comprises NRK-52E cells.

3. Application of a reagent for detecting the expression amount of IL4I1 in the preparation of a kit for judging the effectiveness of a drug in treating renal fibrosis.

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