Application of preparation for inhibiting expression of IL4I1 in preparation of medicine for treating renal fibrosis

Abstract

The invention 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 medicines. In-vivo and in-vitro experiments show that IL4I1 is closely associated with renal injury and renal fibrosis and is a potential target spot for treating renal injury and renal fibrosis. The IL4I1 inhibitor is a substance capable of inhibiting the activity of IL4I1 enzyme, and in-vivo and in-vitro experimental results show that semen plantaginis extract, salted semen plantaginis extract, verbascoside, isoacteoside and akebia quinata phenylethanoid glycoside B are all beneficial to relieving renal fibrosis. Theoretical support is provided for developing drugs for treating renal function impairment and treating and / or relieving renal fibrosis diseases by inhibiting IL4I1.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of a preparation for inhibiting IL4I1 expression in the preparation of a medicine for treating renal fibrosis. Background Art

[0002] Renal fibrosis is a common pathological feature of chronic kidney disease that progresses to the end stage. Its intervention treatment is an important part of preventing and delaying the progression of chronic kidney disease, but there is still a lack of specific treatment measures. Tryptophan metabolism plays an important role in renal fibrosis. Tryptophan can be converted into a series of tryptophan metabolites through host or microbial metabolism, which can activate AHR as a ligand, causing upregulation of protein and transcriptional gene levels, while promoting its entry into the cell nucleus, which in turn triggers the release of downstream signaling molecules and exerts a renal fibrogenic effect.

[0003] Interleukin-4-induced-1 (IL4I1) is a newly discovered tryptophan metabolic enzyme that can metabolize tryptophan into metabolites such as indole-3-lactic acid and indole-3-propionic acid in tumor cells. Currently, most reports on IL4I1 focus on the study of IL4I1's role in tumors and immune regulation. However, there are no reports on the role of IL4I1 in renal fibrosis and whether intervention with IL4I1 can alleviate renal fibrosis. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a preparation for inhibiting the expression of IL4I1 in the preparation of a drug for treating renal fibrosis, so as to solve the problems existing in the above-mentioned prior art. The present invention has found through in vivo and in vitro experiments that IL4I1 is closely related to renal injury and renal fibrosis, and is a potential target for the treatment of renal injury and renal fibrosis. The present invention provides theoretical support for the development of drugs for treating renal damage, treating and / or alleviating renal fibrosis diseases by inhibiting IL4I1.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides application of a preparation for inhibiting IL4I1 expression in preparing a medicine for treating renal fibrosis.

[0007] The present invention also provides the use of IL4I1 as a target in screening drugs for treating renal fibrosis.

[0008] The present invention also provides a method for screening candidate drugs for treating renal fibrosis, comprising subjecting a drug to be screened to a cell model or an animal model of renal fibrosis, and then detecting the expression level of IL4I1 in the cell model or the animal model; if the expression level of IL4I1 is reduced, 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 use of a reagent for detecting IL4I1 expression in preparing a detection kit for judging the effectiveness of a drug in treating renal fibrosis.

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

[0013] The present invention has found through in vitro and in vivo experiments that IL4I1 is closely related to renal injury and renal fibrosis, and is a potential target for the treatment of renal injury and renal fibrosis. IL4I1 inhibitors are a class of substances that can inhibit the expression of IL4I1. The results of in vitro and in vivo experiments show that, such as Plantago extract, salt-processed Plantago extract, verbascoside, isovalascoside, and Akebia phenylethanol glycoside B, are all beneficial to alleviating renal fibrosis.

[0014] The present invention provides theoretical support for developing drugs for treating renal function damage, treating and / or alleviating renal fibrosis diseases by inhibiting IL4I1. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 The protein expression levels of IL4I1 and FN in kidney samples of clinical CKD patients; A is the result of immunofluorescence double staining of IL4I1 and FN; B is the relative expression of IL4I1; C is the relative expression of FN; compared with the normal group, * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001;

[0017] Figure 2 Renal index (A), blood urea nitrogen (B) and blood creatinine biochemical indexes (C) of rats with renal fibrosis model; compared with the normal group, * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001;

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

[0019] Figure 4is the IL4I1 protein content in serum, kidney and NRK-52E cells of rats with renal fibrosis model; A is the IL4I1 content in serum; B is the IL4I1 content in kidney; C is the IL4I1 content in NRK-52E cells; compared with the normal group, * represents P<0.05; ** represents P<0.01; *** represents P<0.001;

[0020] Figure 5 The expression of IL4I1 protein in the kidney of rats with renal fibrosis model; A is the Western Blot detection of IL4I1 protein expression bands after administration of raw and salted plantain; B is the relative quantitative analysis of IL4I1 protein expression; C is the relative quantitative analysis of IL4I1 gene expression by qPCR; RH in B and C represents the raw plantain group, and PH represents the salted plantain group; compared with the blank 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 raw and salted plantain on the expression of IL4I1 in NRK-52E cells; A is the Western Blot detection of IL4I1 protein expression in NRK-52E cells; B is the Western Blot relative quantitative analysis of IL4I1 protein expression in NRK-52E cells; RH in B represents the raw plantain group, and PH represents the salted plantain group; compared with the blank 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 after NRK-52E cells induced renal fibrosis; A is the Western Blot detection of IL4I1 protein expression in NRK-52E cells; B is the Western Blot relative quantitative analysis of IL4I1 protein expression in NRK-52E cells; compared with the blank 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; A is the Western Blot detection of IL4I1 protein expression bands in NRK-52E cells; B is the Western Blot relative quantitative analysis of IL4I1 protein expression in NRK-52E cells; C is the Western Blot relative quantitative analysis of FN protein expression in NRK-52E cells; compared with the WT group, * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001;

[0024] Fig. 9 The expression of renal fibrosis-related transcription factors after IL4I1 overexpression in NRK-52 cells; A is the relative quantitative analysis of IL4I1 gene expression detected by qPCR; B is the relative quantitative analysis of FN gene expression detected by qPCR; C is the relative quantitative analysis of Collagen I gene expression detected by qPCR; D is the relative quantitative analysis of Vimentin gene expression detected by qPCR; E is 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; *** represents P<0.001. DETAILED DESCRIPTION

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

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] Example

[0031] 1. Experimental Materials

[0032] 1. Experimental Animals

[0033] 32 male SPF Sprague Dawley (SD) rats were purchased from Shanghai Slake Laboratory Animal Co., Ltd., weighing 160-180 g. Production license number: SCXK (Shanghai) 2022-0004, and the animal experiment was approved by the Animal Ethics Committee of Zhejiang Chinese Medical University, approval number: IACUC-20220829-12. Before the experiment, the animals were kept in an environment of 23-25 ​​° C, with a 12-hour dark / light cycle. The animals were provided with sufficient food and water during the experiment.

[0034] 2. Drug preparation

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

[0036] Alcohol extracts of raw and salt-roasted plantago seeds: Powder raw and salt-roasted plantago seeds and sieve them, extract them twice with 70% ethanol, reflux and concentrate the extracts, and dry them to obtain extracts. The yield of raw plantago seeds is 4.86%; the yield of salt-roasted plantago seeds is 7.1%. The extracts are dissolved in 0.5% CMC-Na solution, and converted according to the yield of plantago seeds extraction to prepare a 10g / mL raw drug solution.

[0037] 3. Clinical samples

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

[0039] 2. Experimental Methods

[0040] 1. Establishment of animal model of renal fibrosis and grouping and drug administration

[0041] SD male rats were randomly divided into normal group, model group, raw product group, and salt-fried product group, with 8 rats in each group. After one week of adaptive feeding of rats, rats in the model group, raw product group, and salt-fried product group were given 150 mg / kg adenine gavage to establish models. At the same time, the raw product group was also given a raw plantain extract liquid at a dose of 60 g / kg (once a day); the salt-fried product group was given a salt-fried plantain extract liquid at a dose of 60 g / kg (once a day). The rats were weighed and recorded every day during the 28-day experiment, and sufficient normal food and water were provided.

[0042] 2. Serum biochemical index detection

[0043] The levels of serum creatinine (CREA) and blood urea nitrogen (BUN) were detected by automatic biochemical analyzer.

[0044] 3. Kidney tissue staining

[0045] 3.1. Fixing and embedding sections

[0046] (1) Fix the kidney tissue sample with 10% formalin for one week; (2) Remove the kidney tissue from the fixative, cut the kidney tissue with a thickness of about 4 mm, put it in an embedding box, and rinse it with running water for more than 2 hours; (3) 40% ethanol for 20 minutes; (4) 70% ethanol for 15 minutes; (5) 80% ethanol for 10 minutes; (6) 95% ethanol I for 10 minutes; (7) 95% ethanol II for 10 minutes; (8) 100% ethanol I for 10 minutes; (9) 100% ethanol II for 10 minutes; (10) 100% ethanol III for 10 min; (11) Stearic acid: paraffin (1:1; 60°C) for 30 min; (12) Paraffin (60°C) 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, taking care not to shake the tissue. Place the embedded wax block on the freezing table and remove the embedding groove after solidification; (14) Sectioning: Freeze the wax block embedded with the kidney tissue sample in a -20°C refrigerator for more than 40 min before sectioning, and set the section thickness to 4 μm. Place the tissue section in a water bath at 37°C. When the tissue section is flattened, adhere it to the slide, and then place it in an oven at 60°C for 6 hours. When the paraffin on the 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 wax for 2 hours; (2) while still 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 running water 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) seal the sections with neutral gum and observe under an optical microscope.

[0049] 3.3 Masson staining

[0050] The sections were melted and stained according to the instructions of the kit, including Weigert iron hematoxylin staining, acid ethanol differentiation, Masson bluing solution, Ponceau fuchsin staining, weak acid working solution cleaning, phosphomolybdic acid cleaning, aniline blue staining, dehydration, xylene transparentization, neutral gum sealing, and observation under an optical microscope.

[0051] 3.4 Immunofluorescence staining

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

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

[0054] NRK-52E cells in the logarithmic growth phase were seeded in 6-well plates (density 2×10 5 / well), divided into 7 groups, namely blank group, model group, raw plantain group, salt plantain group, verbascoside group, isoverascoside group and akebia phenylethanol glycoside group, see Table 1, each group set 3 replicates. After the cell adhesion state is stable, the serum-free medium is replaced, and the model group is induced by TGF-β1 (10ng / mL) for 24h, and the corresponding drug-containing serum-free medium is added to continue culturing for 24h.

[0055] Table 1 Cell administration groups

[0056]

[0057] 5. Western blot detection

[0058] 5.1. Detection of tissue protein expression

[0059] Extract total tissue protein: Weigh 20 mg of renal tissue cortex, add 100 μL lysis buffer (RIPA:PMSF=100:1, v / v) for every 10 mg of tissue, add magnetic beads, and homogenize the tissue with a pre-cooled tissue cell disruptor until there is no obvious tissue block. Centrifuge at 15000rpm for 10 minutes and take the supernatant. Add 5× Loading buffer (sample: Loading buffer=4:1, v / v) to the protein sample, vortex and mix, put it in a 100℃ metal bath to denature the protein, and store it in a -80℃ refrigerator. After BCA determines the protein concentration of the sample, SDS-PAGE electrophoresis, transfer, block, primary antibody incubation, secondary antibody incubation, development, and detection of target protein expression.

[0060] 5.2. Detection of cell protein expression

[0061] Extract total cell protein: discard the culture medium, add pre-cooled PBS along the wall to wash 3 times, add 1mL PBS solution to each well, collect each group of cell suspension, centrifuge at 4℃5000rpm for 5min, and discard the upper PBS solution. Add 30μl cell lysis solution to the cell pellet and place it on ice, vortex once every 10min, repeat 3 times, then centrifuge at 4℃15000rpm for 15min, take the supernatant and transfer it to a new EP tube for standby use. Take a small amount to measure the concentration, and divide the rest into appropriate amounts and store them at -80℃ to avoid protein degradation caused by repeated freezing and thawing. After measuring the BCA protein concentration of the sample, Western blot is used to detect the expression of the target protein.

[0062] 6. ELISA test

[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 cellular RNA was extracted 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 reference to determine the relative expression level of mRNA.

[0066] 8. CRSPR / Cas9 knockdown of IL4I1

[0067] Design sgIL4I1 primers and synthesize primer sequences (sgIL4I1: gcagcgtccgaccacgtat, SEQ ID NO.1). PCR amplification, ligation, transformation, and shake amplification are then spread on plates containing ampicillin resistance and cultured for 12-16 hours. Select a single colony and amplify it; extract the plasmid according to the instructions of the endotoxin-free plasmid extraction kit and sequence it. The plasmid with the correct sequencing is the constructed IL4I1 knockout plasmid.

[0068] 293T cell packaging lentivirus: Use DMEM medium without double antibodies to plate 293T, and the confluence of 293T reaches 90-100%. Configure the transfection complex according to the following system: Add 1200ng VSVG, 2400ng PSPA, and 4800ng target plasmid to 0.5mL serum-free and antibiotic-free opti-MEM medium. After pipetting and mixing, add 18μL of infection-aiding reagent PEI 40000, pipet and mix again, and incubate at room temperature for 20min. Then, the transfection complex is evenly dripped into the 293T cells and cultured in a 37°C, 5% CO2 incubator for 8h. After 8h, replace it with ordinary DMEM complete medium. After 48h, collect the supernatant and add 5X PEG8000 (the ratio of medium: PEG8000 is 4:1) to concentrate overnight. Centrifuge at 4°C, 4800 rpm for 10 min, discard the supernatant, resuspend the virus in 400 μL PBS, aliquot, and store at -80°C.

[0069] Lentivirus infection of NRK-52E cells: NRK-52E cells were plated in 24-well plates with DMEM medium without dual antibodies. On the second day, the infection enhancer polybrene was added to the medium at a ratio of 1:1000, and 400μL of medium and 100μL of lentivirus were added to each well. After 8 hours, it was replaced with ordinary DMEM complete medium, and after it was full, it was inoculated into a 6-well plate or T25, and 1ng / mL of puromycin was used to continuously kill the cells, leaving the successfully transfected cells, and subsequent WB verification was performed to obtain a NRK-52E cell line with stable knockout of IL4I1.

[0070] 9. IL4I1 Overexpression

[0071] The rat IL4I1 cDNA clone plasmid was ordered from the company, and then 293T cells were used to package lentivirus. The lentivirus was used to infect NRK-52E cells and the operation was the same as that of the knockout cell line construction.

[0072] 3. Results

[0073] 1. IL4I1 protein is highly expressed in the kidneys of CKD patients

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

[0075] Table 2 Statistics of mean fluorescence intensity in immunofluorescence

[0076]

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

[0078] The rats in the normal group had the characteristics of good eating, agile movement, normal urination and defecation. After intragastric administration of adenine, the rats began to show mental depression, slow reaction, often curled up, weight loss, thirst, increased urine volume, and soft stools that easily caused the bedding to become wet. At the beginning of the experiment, there was no significant difference in the initial weight of the rats in each group. Table 3 and Figure 2 The results show the renal index, serum urea nitrogen, serum creatinine and serum IL4I1 content of rats. Figure 2 As shown, compared with the normal group, the levels of renal index, creatinine and urea nitrogen in the serum of rats in the model group were significantly increased (***P<0.001).

[0079] Table 3 Kidney index, serum urea nitrogen (BUN), serum creatinine (CARE), serum IL4I1 content in rats

[0080]

[0081]

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

[0083] Kidney pathological sections HE staining Figure 3 As shown in the figure, the kidney structure of the rats in the normal group was clear and tightly arranged, and no abnormalities were found in the renal tubules, glomeruli and renal interstitium. The renal tissue structure of the model group was disordered, with vacuoles formed, a large amount of brown-yellow 2,8-dihydroxyadenine deposits were seen in the renal tubules, the number of glomeruli decreased and atrophied, and the renal tubules were dilated and irregularly arranged. According to the results of Masson staining, there was a small amount of fibrous tissue around the glomeruli in the normal group, and there was obvious blue collagen deposition around the glomeruli and renal tubules in the model group, forming renal interstitial fibrosis.

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

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

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

[0087]

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

[0089] The expression of IL4I1 protein level and transcription level of raw PS and Pro PS in adenine-induced renal fibrosis model rats was detected by WB and qPC. After statistical analysis, see Table 5 and Figure 5 Compared with the blank group, the protein level and transcription level of IL4I1 in the model group increased significantly, while raw plantain reduced the protein level of IL4I1 in the model, and salt plantain significantly reduced the protein level and transcription level of IL4I1 in the model, indicating that salt plantain is better than raw plantain in reducing IL4I1 in the renal fibrosis model.

[0090] Table 5 WB detection of IL4I1 protein and mRNA levels in rat kidneys after administration of Plantago asiatica (n=8)

[0091]

[0092]

[0093] 6. Western Blot detection of IL4I1 protein expression in NRK-52E cells in the TGF-β-induced renal fibrosis model group and treatment group

[0094] TGF-β induced renal fibrosis in NRK-52E cells, and administration of Raw PS and Pro PS alleviated renal fibrosis. WB was used to detect the changes in the protein expression level of IL4I1 in the renal fibrosis model after administration of Pro PS. It was found that after NRK-52E cells were induced to renal fibrosis by TGF-β, the protein level of IL4I1 increased significantly, and renal fibrosis could be alleviated after administration, and the protein level of IL4I1 decreased, as shown in Table 6 and Figure 6 shown.

[0095] Table 6 WB determination of IL4I1 protein expression in NRK-52E cells after administration of Plantago

[0096]

[0097] 7. Western Blot detection of IL4I1 protein expression in TGF-β-induced NRK-52E cell renal fibrosis model group and verbascoside, isovalascoside, and ketophenone glycoside B treatment groups

[0098] The changes in the protein expression level of IL4I1 in the renal fibrosis model after administration of Cal B, Iso and Ace were detected by WB. It was found that after NRK-52E cells were induced to renal fibrosis by TGF-β, the protein level of IL4I1 was significantly increased, while the administration of Cal B, Iso and Ace could alleviate renal fibrosis and the protein level of IL4I1 was reduced, as shown in Table 7 and Figure 7 shown.

[0099] Table 7 Expression of IL4I1 protein in NRK-52E cells after administration of Akebia trifoliata phenylethanol glycoside B, Isocorcinum, and Calycosus

[0100]

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

[0102] After knocking down IL4I1 in NRK-52E cells, it was found that the expression of renal fibrosis-related proteins also decreased. After the cells were stimulated with TGF-β to establish a model, the expression of renal fibrosis-related proteins and IL4I1 proteins in the non-knocked-down cells (WT) increased significantly, while the cells with knocked-down IL4I1 (KO) did not cause the expression of fibrosis-related proteins after TGF-β stimulation, as shown in Table 8 and Figure 8 This indicates that IL4I1 is closely related to renal fibrosis and can be used as a potential target for the treatment of renal fibrosis.

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

[0104]

[0105] 9. qPCR detection of the expression of renal fibrosis-related transcription factors after NRK-52 cells overexpressed IL4I1

[0106] In NRK-52E cells, after IL4I1 was overexpressed, the transcriptional levels of renal fibrosis-related factors also increased, as shown in Tables 9 and Fig. 9 , further indicating that IL4I1 is associated with renal fibrosis.

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

[0108]

[0109] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a preparation that inhibits IL4I1 expression in the preparation of a drug for treating renal fibrosis.

2. Application of IL4I1 as a target in screening drugs for the treatment of renal fibrosis.

3. A method for screening candidate drugs for treating renal fibrosis, characterized in that: The method comprises subjecting the drug to be screened to a cell model or an animal model of renal fibrosis, and then detecting the expression level of IL4I1 in the cell model or the animal model. If the expression level of IL4I1 is reduced, the drug to be screened is a candidate drug for treating renal fibrosis.

4. The method according to claim 3, characterized in that The cell model is a kidney cell model.

5. The method according to claim 4, characterized in that The kidney cells include NRK-52E cells.

6. Application of reagents for detecting IL4I1 expression in the preparation of detection kits for judging the effectiveness of drug treatment for renal fibrosis.

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