Use of lipocalin 2 in the preparation of a formulation for the diagnosis or treatment of peritoneal dysfunction

Abstract

The present application relates to the application of lipocalin 2 in the preparation of preparations for diagnosing or treating peritoneal dysfunction. The present application excavates markers for peritoneal dysfunction diagnosis and treatment, detects the expression changes of LCN2 in the peritoneal tissue and effusion of peritoneal dialysis patients and peritoneal dysfunction model mice, determines the relationship between LCN2 changes and acquired peritoneal dysfunction, observes the expression changes of LCN2 and its functional effects on inflammatory fibrosis, and further constructs Lcn2 Gene knockout or knockdown expression mice, and found that knocking out Lcn2 The expression level of the gene or knocking it down can effectively alleviate the pathological phenotype of peritoneal dysfunction, indicating that LCN2 can be applied to identify peritoneal dysfunction of peritoneal dialysis patients, and develop a target for treating peritoneal dialysis related peritoneal dysfunction drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of lipid transporter 2 in the preparation of formulations for the diagnosis or treatment of peritoneal dysfunction. Background Technology

[0002] Lipocalin 2 (LCN2) is a secreted cytokine consisting of a β-barrel structure composed of eight antiparallel β-sheets. Traditionally, it was thought to originate primarily from immune cells such as neutrophils and macrophages, but recent studies have found it is also expressed in various epithelial and parenchymal cells (such as hepatocytes, renal tubular epithelial cells, and adipocytes). Human LCN2 protein exists in three secretory forms: a 25 kDa monomer, a 46 kDa homodimer, and a 135 kDa heterocomplex formed with matrix metalloproteinase 9, and it can interact with multiple receptors. Previous studies have shown that LCN2 is closely related to the development and progression of various diseases, including cancer, kidney injury, Parkinson's disease, and hepatitis. Furthermore, numerous studies have confirmed that LCN2 can serve as a biomarker for diseases such as non-alcoholic steatohepatitis, renal tubular injury, and sepsis.

[0003] For example, CN118011013A discloses the application of the biomarker LCN2 in the diagnosis and screening of sepsis-associated encephalopathy (SAE). Using proteomics, it was the first time that LCN2 could be used for early screening of SAE. By detecting the expression level of LCN2 in human plasma, the occurrence of SAE can be effectively predicted. In this invention, when using the biomarker LCN2 for early screening of SAE, the concentration of LCN2 in SAE patients and healthy individuals was semi-quantitatively detected using enzyme-linked reaction (ELISA), showing a significant difference between the two groups (p < 0.05). This screening method also provides an important detection tool for long-term tracking of high-risk groups for SAE, facilitating early detection and significantly reducing patient mortality.

[0004] Acquired peritoneal dysfunction is not a standalone disease, but rather an irreversible damage to the structure and function of the peritoneum caused by various factors during long-term peritoneal dialysis (PD). These factors include the use of bioincompatible peritoneal dialysis solutions, recurrent peritonitis, decreased residual renal function, and increased dialysis duration. The core manifestation is inadequate ultrafiltration. Although LCN2 can serve as a biomarker for various diseases and is involved in their development, its role in peritoneal dysfunction in dialysis patients has not been reported.

[0005] In conclusion, the development of diagnostic and therapeutic biomarkers for peritoneal dysfunction in peritoneal dialysis patients is of great significance. Summary of the Invention

[0006] In response to the shortcomings of existing technologies and practical needs, this invention provides the application of lipid transporter 2 in the preparation of formulations for the diagnosis or treatment of peritoneal dysfunction, aiming to provide new uses for lipid transporter 2 and promote the development of the field of diagnosis and treatment of peritoneal dysfunction in peritoneal dialysis patients.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a biomarker associated with peritoneal dysfunction, the biomarker including lipid transporter 2.

[0008] In this invention, it was found that the expression level of lipid transporter 2 was significantly increased in individuals with peritoneal dialysis-related peritoneal dysfunction, and it has a certain degree of specificity, which can be effectively used to identify peritoneal dysfunction in peritoneal dialysis patients.

[0009] In a second aspect, the present invention provides the use of lipid transporter 2 and / or its detection reagents in the preparation of products for detecting peritoneal dysfunction.

[0010] Optionally, the detection reagent includes a reagent for detecting the expression level of lipid transporter 2.

[0011] Thirdly, the present invention provides a kit for detecting peritoneal dysfunction, the kit comprising reagents for detecting the expression level of lipid transporter 2.

[0012] Fourthly, the present invention provides the application of lipid transporter 2 and / or its detection reagents in screening interventions for peritoneal dysfunction.

[0013] In this invention, it was found that knocking out the lipid transporter 2 gene or knocking down its expression level in a peritoneal dysfunction model can effectively alleviate peritoneal dysfunction-related pathological phenotypes, indicating that lipid transporter 2 can serve as a potential therapeutic target for peritoneal dysfunction.

[0014] Fifthly, the present invention provides a method for screening interventions for peritoneal dysfunction, the method comprising: administering a candidate intervention to an individual with peritoneal dysfunction and comparing the changes in the expression level of lipotransferase 2 in the candidate intervention-treated group and the untreated control group; when the candidate intervention reduces the expression level of lipotransferase 2, the candidate intervention is determined to have the potential to intervene in peritoneal dysfunction.

[0015] Optionally, the candidate intervention includes a gene-editing agent for lipid transporter 2.

[0016] Optionally, the gene editing reagent for lipid transporter 2 includes a reagent that knocks out the gene encoding lipid transporter 2 or knocks down its expression level.

[0017] Optionally, the peritoneal dysfunction individuals include animal models of peritoneal dysfunction (such as mouse models).

[0018] In a sixth aspect, the present invention provides the application of a gene-editing reagent for lipid transporter 2 in the preparation of an intervention for peritoneal dysfunction, wherein the gene-editing reagent comprises a reagent that knocks out the gene encoding lipid transporter 2 or knocks down its expression level.

[0019] In a seventh aspect, the present invention provides an intervention for peritoneal dysfunction, the intervention comprising a reagent that knocks out the gene encoding lipid transporter 2 or knocks down its expression level.

[0020] Compared with the prior art, the present invention has at least the following beneficial effects: This invention provides an in-depth analysis of the mechanisms related to peritoneal dysfunction in peritoneal dialysis patients, identifies biomarkers for diagnosis and treatment, and detects changes in LCN2 expression in peritoneal tissue and dialysis fluid of peritoneal dialysis patients and mouse models of peritoneal dysfunction. It clarifies the relationship between LCN2 changes and acquired peritoneal dysfunction, observes changes in LCN2 expression and its functional role in inflammation and fibrosis, and further constructs a mouse model of peritoneal dysfunction. Lcn2 Gene knockout mice, and mice carrying sh via intraperitoneal injection. Lcn2 Adeno-associated virus 2-mediated peritoneal localization Lcn2 Decreased gene expression levels were observed in a peritoneal dysfunction model when the gene was knocked out. Lcn2 Both gene knockdown and knockdown of its expression level can effectively alleviate peritoneal dysfunction-related pathological phenotypes, indicating that LCN2 can be used to identify peritoneal dysfunction in peritoneal dialysis patients and as a target for developing drugs to treat peritoneal dialysis-related peritoneal dysfunction. Attached Figure Description

[0021] Figure 1 The images show the results of LCN2 expression in peritoneal dialysis fluid samples. Figure A shows the expression of LCN2 in exfoliated cells of dialysis fluid from patients with short dialysis duration (n = 4) and long dialysis duration (n = 4) by Western blotting, and the quantitative analysis of the relative levels of LCN2 / β-actin. Figure B shows the concentration of LCN2 in peritoneal dialysis fluid from patients with dialysis duration <6 months (n = 23), 6-12 months (n = 11), and >12 months (n = 14) by ELISA.

[0022] Figure 2The graph shows the correlation between LCN2 concentration in the percolation fluid of PD patients and PET results. In the graph, LCN2 concentration in the percolation fluid is negatively correlated with ultrafiltration volume (n = 48), LCN2 concentration in the percolation fluid is negatively correlated with D / D0 glucose ratio (n = 48), and LCN2 concentration in the percolation fluid is positively correlated with 4-h D / Pcr (n = 48).

[0023] Figure 3 ROC curves for identifying rapid PSTRs using human dialysis fluid LCN2 concentration.

[0024] Figure 4 Figure A shows the expression characteristics and changes of LCN2 in animal models. Figure A shows the expression of LCN2 in the visceral peritoneum of mice detected by Western blotting and the quantitative analysis of the relative levels of LCN2 / GAPDH (n = 6 per group). Figure B shows the expression of LCN2 in the visceral peritoneum of mice detected by qPCR (n = 6 per group). Figure C shows the expression of LCN2 in the parietal peritoneum of mice detected by immunohistochemistry and the number of LCN2-positive cells per high-power field (n = 6 per group). ; ; ; .

[0025] Figure 5 The graph shows the correlation between LCN2 expression in mouse peritoneum and PET results and pathological features. In the graph, A shows a negative correlation between LCN2 expression in peritoneum and the D / D0 glucose ratio (n = 17); B shows a positive correlation between LCN2 expression in peritoneum and peritoneal thickness (n = 18); and C shows a positive correlation between LCN2 expression in peritoneum and the number of peritoneal vessels (n = 18).

[0026] Figure 6A Wild type and Lcn2 - / - Representative images of Masson staining and hematoxylin-eosin (HE) staining in peritoneal sections of mice (treated with CG or saline as controls), and quantitative data on peritoneal thickness (n = 5).

[0027] Figure 6B Wild type and Lcn2 - / - Ultrafiltration data of mice (n=5) (treated with CG or saline as controls).

[0028] Figure 6C Wild type and Lcn2 - / - Data on the glucose ratio of dialysate at 2 hours to dialysate at 0 hours in mice (n = 5) (treated with CG or saline as controls).

[0029] Figure 6D Wild type and Lcn2 - / - Data on the dialysate to plasma (D / P) urea ratio in mice (n = 5) (treated with CG or saline as controls).

[0030] Figure 6E Wild type and Lcn2 - / - Representative images and quantitative data of CD31 and VEGF-A immunostaining in peritoneal sections of mice (treated with CG or saline as controls).

[0031] Figure 6F Wild type and Lcn2 - / - Representative images and quantitative data of F4 / 80 immunostaining in peritoneal sections of mice (n = 5) (treated with CG or saline as controls).

[0032] Figure 6G Wild type and Lcn2 - / - Peritoneum of mice (treated with CG or saline as controls) Tnf and Il-6 RT-qPCR results of relative mRNA levels (n = 5).

[0033] Figure 6H Representative images and quantitative data for Fibronectin and Colleagen I immunostaining (n = 5).

[0034] Figure 6I Wild type and Lcn2 - / - Representative immunoblot images and quantitative data of Fibronectin, Colleagen I, α-SMA and LCN2 in the peritoneum of mice after treatment with CG or saline (n = 5 per group).

[0035] Figure 7A Masson staining and hematoxylin-eosin staining in AAV2-sh Lcn2 Representative images from peritoneal sections of the treatment group and AAV2-control (induced by CG or control), and quantitative data on peritoneal thickness (n = 5).

[0036] Figure 7B AAV2-sh induced by CG or control Lcn2Data on the D / D0 glucose ratio of mice in the treatment group and the AAV2-control group (n = 5).

[0037] Figure 7C AAV2-sh induced by CG or control Lcn2 Data on the D / P urea ratio of mice in the treatment group and the AAV2-control group (n = 5).

[0038] Figure 7D AAV2-sh induced by CG or control Lcn2 Representative images and quantitative data of CD31 and VEGF-A immunostaining in peritoneal sections of mice in the treatment and AAV2-control groups (n = 5).

[0039] Figure 7E AAV2-sh induced by CG or control Lcn2 Representative images and quantitative data of F4 / 80 immunostaining in peritoneal sections of mice in the treatment and AAV2-control groups (n = 5).

[0040] Figure 7F AAV2-sh induced by CG or control Lcn2 In the peritoneum of treatment group and AAV2-control mice Il1b and Il6 RT-qPCR results of relative mRNA levels (n = 5).

[0041] Figure 7G For AAV2-sh Lcn2 Representative images and quantitative data of Fibronectin I and Colleagen I immunostaining in mice in the treatment and AAV2-control groups (n = 5).

[0042] Figure 7H Representative immunoblot patterns and the presence of Collegen I, α-SMA, and LCN2 in AAV2-sh Lcn2 Quantitative data in the peritoneum of treatment and AAV2-control mice induced by CG or control (n = 5 per group). Detailed Implementation

[0043] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0044] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0045] This invention delves into the mechanisms of peritoneal dysfunction in peritoneal dialysis patients, aiming to identify biomarkers for diagnosis and treatment. Specifically, it detects changes in the expression of lipid carrier protein 2 (LCN2) in peritoneal tissue and dialysis fluid of peritoneal dialysis (PD) patients and mouse models of peritoneal dysfunction, and analyzes its correlation with clinical indicators of peritoneal function to clarify the relationship between LCN2 changes and acquired peritoneal dysfunction. An in vitro model of inflammatory fibrotic peritoneal mesothelial cells is constructed, and changes in LCN2 expression and its functional role in inflammatory fibrosis are observed. Furthermore, based on the mouse model of peritoneal dysfunction, a... Lcn2 Gene knockout mice ( Lcn2 - / - ), and via intraperitoneal injection carrying sh Lcn2 Adeno-associated virus 2 (AAV2-sh Lcn2 Local peritoneal mediated by ) Lcn2 Gene knockdown was used to investigate the role of LCN2 in regulating key pathological changes such as peritoneal inflammation, angiogenesis, and fibrosis, in order to elucidate the role of LCN2 in acquired peritoneal dysfunction.

[0046] Example 1 This embodiment examines changes in LCN2 in PD patients and mice with peritoneal dysfunction.

[0047] Nocturnal peritoneal dialysis fluid and exfoliated cells were collected from 48 patients with peripheral dysplasia (PD). LCN2 expression levels were detected using enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB). The correlation between LCN2 concentration in the dialysis fluid and dialysis duration and peritoneal transport function was analyzed, and the predictive efficacy of LCN2 for acquired peritoneal dysfunction was evaluated. The specific experimental procedures included: Processing of peritoneal dialysis fluid specimens from PD patients: (1) Collect overnight dialysis fluid from PD patients within 1 hour of extubation and centrifuge at 500g for 10 min; (2) Take the supernatant of the dialysis fluid for enzyme-linked immunosorbent assay (ELISA); (3) Perform subsequent immunoblotting analysis on cell precipitation proteins. Patients were divided into short-term PD patients (dialysis duration <6 months) and long-term PD patients (dialysis duration >36 months) based on their dialysis duration.

[0048] ELISA detection of PD patient transdermal fluid: (1) Prepare transdermal fluid samples according to the above processing method; (2) Warm the human LCN2 kit (BY-EH111771) to room temperature for 60 min, and prepare the working solutions of each component of the kit according to the instructions. (3) Set up standard wells, blank wells and sample wells. Add 50 μL of different concentrations of standard to each standard well, 50 μL of sample diluent to each blank well, and 50 μL of transdermal fluid sample to each sample well. (4) Add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to all wells. (5) Cover the reaction plate with sealing film and incubate at 37℃ in the dark for 60 min. (6) Remove the sealing film, discard the liquid, pat dry on absorbent paper, add 200 μL of washing buffer to each well, let stand for 20 s, shake off the washing buffer, pat dry on absorbent paper, and repeat this process 5 times. (7) Mix substrates A and B thoroughly at a 1:1 volume ratio, and add 100 μL of the substrate mixture to all wells. Cover the reaction plate with sealing film and incubate at 37°C in the dark for 15 min. (8) Add 50 μL of stop solution and read the absorbance (OD value) of each well on a 450 nm wavelength microplate reader. (9) Plot the standard concentration as the x-axis and the corresponding OD value as the y-axis, and use a four-parameter Logistic curve fitting to create a standard curve equation. Calculate the concentration value of each permeate sample using its OD value.

[0049] PD patient transudate cell pellet WB detection: (1) Prepare 1×RIPA protein lysis buffer: 1 mL of 1×RIPA protein lysis buffer is diluted with 9 mL of double distilled water (ddH2O), and 1 tablet of protease inhibitor and 1 tablet of phosphatase inhibitor are added. After dissolving and mixing, it is placed on ice for pre-cooling; (2) Extract protein: 500 μL of 1×RIPA protein lysis buffer is added to the extracted cell pellet, and after resuspending by pipetting, it is placed on ice for 15 min; (3) Sonication: 20% power, sonication for 5s, interval of 5s, repeated twice; (4) Centrifugation: 4℃, 12000 rmp, 15 min; (5) Obtain protein supernatant: the colorless and clear supernatant of the uppermost layer is transferred to a new EP tube and placed on ice; (6) BCA method to determine protein concentration: according to the description of the BCA kit, the corresponding volume of protein supernatant and solutions A and B are added to a 96-well plate and incubated in a 37℃ incubator for 30 minutes. (7) Balancing: According to the protein concentration of each sample, add an appropriate amount of 5× Loading Buffer and ddH2O to balance; (8) Protein denaturation: Place the balanced protein in a metal bath and heat at 100℃ for 15 min to denature the protein and obtain a stable protein sample; (9) Electrophoresis: Use MOPS-SDS buffer as electrophoresis solution, load 20 μL of each sample, and gradually increase the voltage from 65V to 120V; (10) Preparation of transfer solution: Add 200 mL of methanol and 100 mL of 10× transfer buffer to 700 mL of ddH2O to obtain 1× transfer solution, and pre-cool on ice; (11) Transfer: Transfer at 100V constant voltage for 90 min; (12) Blocking: Use TBS to prepare 5% skim milk as blocking solution, block at room temperature for 1 h, and use TBS shaker for 5 min. (13) Primary antibody preparation: Prepare the corresponding primary antibody using primary antibody dilution buffer, specifically as follows: human LCN2 antibody (1:1000); GAPDH-HRP antibody (1:5000); (14) Primary antibody incubation: Incubate overnight at 4℃; (15) Washing: Wash on a shaker for 5 min × 3 times using TBS-T; (16) Secondary antibody preparation: Prepare the corresponding secondary antibody using primary antibody dilution buffer (1:5000); (17) Secondary antibody incubation: Incubate at room temperature for 90 min; (18) Washing: Wash on a shaker for 5 min × 3 times using TBS-T; (19) Exposure: Add an appropriate amount of ECL luminescent solution for exposure; (20) Data analysis: Calculate the relative expression level using ImageJ software based on the gray value.

[0050] Experimental results are as follows Figure 1As shown in Figure A, Western blotting (WB) reveals the expression of LCN2 in exfoliated cells from the dialysis fluid of patients in the short dialysis period group (n = 4) and the quantitative analysis of the relative levels of LCN2 / β-actin; Figure B shows the concentration of LCN2 in the peritoneal dialysis fluid of patients in the dialysis period <6 months (n = 23), 6-12 months (n = 11), and >12 months (n = 14) groups detected by ELISA. ; ELISA and Western blot analysis of dialysate from PD patients showed significantly elevated LCN2 expression in patients with long dialysis histories. The LCN2 concentration in the dialysate was negatively correlated with ultrafiltration volume and the D / D0 glucose ratio, and positively correlated with the 4-hour D / Pcr (4-hour dialysate to plasma creatinine ratio). Figure 2 In addition, representative images of masson staining and hematoxylin-eosin (HE) staining in peritoneal sections from wild-type and Lcn2- / - mice (treated with CG or saline controls), along with quantitative data on peritoneal thickness (n = 5), were used. The area under the ROC curve (AUC) for identifying rapid PSTRs using peritoneal dialysis fluid LCN2 concentration reached 0.9. The AUC for identifying rapid peritoneal transport rates using dialysis fluid LCN2 concentration reached 0.9 (…). Figure 3 ).

[0051] A mouse model of peritoneal dysfunction was established using two methods: intraperitoneal injection of chlorhexidine glucose (CG) and 4.25% glucose dialysis fluid + LPS (PDS). Immunohistochemistry, Western blotting, and qPCR were used to detect LCN2 expression in the peritoneum of these mice, and its correlation with peritoneal transport rate, peritoneal thickness, and the number of neovascularizations was analyzed. The specific experimental procedures included: Establishment of mouse models of acquired peritoneal dysfunction: (1) High glucose peritoneal dialysis solution + LPS (PD dialysate plus LPS, PDS) model: Male mice were injected intraperitoneally with 3 mL of 4.25% glucose dialysis solution (once daily) and LPS 1 mg / kg (three times a week) was added to the dialysis solution; the mice were killed after 6 weeks of administration. (2) Chlorhexidine gluconate (CG) model: Male mice were injected intraperitoneally with 10 ml / kg of 0.1% CG (every other day); the mice were killed after 2 weeks of administration.

[0052] Mouse PET: (1) Intraperitoneal injection of 3 mL of 4.25% glucose peritoneal dialysis solution into mice, followed by 2 h of peritoneal retention; (2) Collection of dialysis fluid and serum samples from mice after 2 h; (3) Detection of glucose, creatinine, and urea nitrogen concentrations in dialysis fluid and serum samples from mice; (4) Calculation of ultrafiltration volume as the difference between the volume of dialysis fluid drawn out in 2 h and the volume of fluid injected in 3 mL; (5) Calculation of D / D0 glucose ratio as glucose concentration of dialysis fluid in 2 h / glucose concentration of injected peritoneal dialysis fluid; (6) Calculation of D / Pcr ratio as creatinine concentration of dialysis fluid in 2 h / serum creatinine concentration; (7) Calculation of D / Pur ratio as urea nitrogen concentration of dialysis fluid in 2 h / serum urea nitrogen concentration.

[0053] Mouse sample collection: (1) Anesthetize mice by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg); (2) Collect blood by enucleation, collect blood in 1.5 mL EP tubes, let stand at room temperature, centrifuge at 3000 rpm for 15 min at room temperature, collect the supernatant in 1.5 mL EP tubes, and store in a -80℃ refrigerator; (3) Wipe with 75% alcohol cotton balls after blood collection. Subsequently, a 1cm incision was made below the xiphoid process of the mouse, and the syringe was inserted into the peritoneal cavity through the incision to draw out the transudate. The volume of the transudate was recorded and collected in a centrifuge tube. The tube was centrifuged at 4℃ and 600g for 15 min. The supernatant was then collected in a 1.5 mL EP tube and stored in a -80℃ freezer. (4) After collecting the transudate, the mouse was fixed in a supine position. The skin was carefully separated from the abdominal wall of the mouse. After avoiding the injection site of the drug in the peritoneal cavity, the abdominal wall was cut off and placed in 4% paraformaldehyde. It was then fixed in a 4℃ freezer for 24 h before being embedded in paraffin and sectioned. (5) The visceral peritoneal tissue of the mouse was placed in a cryovial, quickly frozen in liquid nitrogen, and then transferred to a -80℃ freezer for storage.

[0054] Hematoxylin-Eosin (HE) staining of paraffin sections of mouse parietal peritoneum: (1) Baking: Take 4 μm thick paraffin sections and bake them in a constant temperature drying oven at 65℃ for 1 h; (2) Dewaxing to hydration: Xylene I 15 min → Xylene II 15 min → Anhydrous ethanol I 10 min → Anhydrous ethanol II 10 min → 95% ethanol 5 min → 90% ethanol 5 min → 80% ethanol 5 min → 75% ethanol 5 min → ddH2O 5 min; (3) Nuclear staining: Use hematoxylin after filtration and stain for 8 min; (4) Blueing: Rinse with tap water and observe under a microscope until the nuclei turn blue and the cytoplasm is colorless, about 1 h; (5) Cytoplasmic staining: Stain with eosin for 2 min until the cytoplasm is pink; (6) Washing: Pat dry on a paper towel and then rinse with ddH2O; (7) Dehydration: 75% ethanol 5 min → 80% ethanol 5 min → ddH2O 5 min min→90% ethanol 5 min→95% ethanol 5 min Anhydrous ethanol I 5 min→Anhydrous ethanol II 5 min→Xylene I 15 min→Xylene II 15 min; (8) Mounting: Add neutral resin to mount the slide; (9) Slide scanning: Use a pathological slide scanner to scan and store the slide.

[0055] Masson staining of paraffin sections of mouse parietal peritoneum: (1) Baking and dewaxing to hydration are the same as above; (2) Prepare the corresponding reagents according to the Masson staining kit; (3) Immerse the sections in Bouin's Solution overnight at room temperature; (4) Rinse with tap water until the sections are colorless, about 1 hour; (5) Stain in Weigert's Iron Hematoxylin Solution for 5 minutes; (6) Rinse with tap water for 5 minutes, then rinse with ddH2O; (7) Stain in Biebrich Scarlet-Acid Fucshin for 5 minutes; (8) Rinse with ddH2O; (9) Stain in Phospho-ungstic / Phosphomolybdic Acid Solution for 5 minutes; (10) Stain in Aniline Blue Solution for 5 minutes; (11) Stain in 1% glacial acetic acid for 2 minutes; (12) Dehydration, mounting and scanning are the same as above.

[0056] Immunohistochemical staining of paraffin sections of mouse parietal peritoneum: (1) Baking and dewaxing to hydration are the same as above; (2) Antigen retrieval: High-pressure retrieval is performed using 1× sodium citrate buffer (10 min, 121℃), followed by natural cooling overnight, and PBS washing for 5 min × 3 times; (3) Draw a circle around the tissue using a histochemical pen; (4) Punching: Punch 50 μL of 0.2% Triton X-100 for 5 min, followed by PBS washing for 5 min × 3 times; (5) Inactivation of endogenous catalase in the tissue section: Add 1 drop of 3% hydrogen peroxide (about 50 μL, just enough to cover the tissue), incubate in a humidified chamber at room temperature for 10 min, and then wash with PBS for 5 min × 3 times; (6) Blocking: Add 50 μL of 5% BSA blocking solution, incubate in a humidified chamber at room temperature for 60 min; (7) Primary antibody preparation: Use 1% BSA blocking solution. BSA was used to prepare the primary antibody as follows: mouse LCN2 antibody (1:500); mouse CD31 antibody (1:2000); mouse VEGFA antibody (1:100); mouse Fibronectin antibody (1:200); mouse Colleagen I antibody (1:500); mouse CD3 antibody (1:1000); mouse F4 / 80 antibody (1:400); (8) Primary antibody incubation: Remove the blocking solution, add 50 μL of specific antibody, incubate overnight at 4°C in a humidified chamber, and then rinse with PBS for 5 min × 3 times; (9) Secondary antibody incubation: Add 50 μL of secondary antibody from the mouse / rabbit polymer immunohistochemistry detection system, incubate at room temperature in a humidified chamber for 1 h, and then rinse with PBS for 5 min × 3 times; (10) DAB staining: Place the slide under a microscope and add 50 μL of freshly prepared DAB. The solution is developed (A solution is protected from light: B solution = 1:20), and the reaction time is controlled under a microscope, about 30s-3min; (11) Terminate the reaction: after rinsing several times in a graduated cylinder containing 1 L ddH2O, soak in ddH2O to terminate the reaction, soak in ddH2O for 2 min × 3 times; (12) Counterstain the cell nuclei: counterstain the cell nuclei with hematoxylin stock solution for 30s; (13) Rinse and return to blue: rinse the slide with running tap water for 1 minute; (14) Dehydration, mounting and scanning are the same as above.

[0057] Quantitative real-time PCR (qPCR) of mouse visceral peritoneum: (1) Tissue sample lysis: Visceral peritoneum tissue was excised on ice and placed in a grinding tube containing 1 mL Trizol. Two 3 mm grinding beads were added and the sample was homogenized thoroughly using a homogenizer (specific parameters: 60 hz, 10 s interval every 30 s, 3 cycles). After homogenization, the sample was allowed to stand on ice for 10 min. After removing the grinding beads, the lysis buffer was transferred to an RNase-free EP tube. (2) 200 μL of chloroform substitute was added and the sample was vigorously shaken to mix for about 30 s. The sample was allowed to stand at room temperature for 5 min. (3) Centrifugation: 4℃, 12000 rpm, 15 min. (4) Collection of supernatant: The colorless layer was carefully aspirated, avoiding the middle milky white layer. About 400 μL of the supernatant was placed in a new RNase-free EP tube. (5) An equal volume of isopropanol was added and the sample was gently inverted to mix. The sample was allowed to stand at room temperature for 15 min. (6) Centrifugation: 4℃, 12000 rpm, 10 min. (7) Discard the supernatant and keep the precipitate; (8) Wash the precipitate: add 500 μL of pre-cooled 75% ethanol (prepared with DEPC water and anhydrous ethanol); (9) Centrifuge: 4℃, 12000 rpm, 3 min; (10) Discard the supernatant and invert it onto absorbent paper to dry the liquid; (11) Repeat steps 8-10; (12) Let stand in a fume hood for 10 min; (13) Add 50 μL of DEPC water to dissolve the RNA and mix by pipetting. After dissolving, place it on ice for temporary storage; (14) Measure the RNA concentration: use a NanoDrop 2000 UV spectrophotometer at a detection wavelength of 260 / 280 nm, with DEPC water as a blank control, and use spectrophotometry to determine the concentration and purity of RNA; (15) Perform reverse transcription and qPCR according to the kit instructions; (18) Data analysis: calculate the corresponding relative expression level based on the Ct value.

[0058] WB of mouse visceral peritoneum: (1) Protein extraction from visceral peritoneum: 50 mg of tissue was placed in a grinding tube containing 500 µL 1×RIPA, and 2 3 mm grinding beads were added. The sample was homogenized thoroughly using a homogenizer (specific parameters: 60 hz, 10 s interval every 30 s, 3 cycles). After homogenization, the grinding beads were removed and the lysate was transferred to a new EP tube; (2) Ultrasonic disruption: 20% power, ultrasonic for 5 s, 5 s interval, repeated twice; (3) Centrifugation: 37℃, 12000 rmp, 15 min; (4) Obtain protein supernatant: Transfer the top layer of colorless and clear supernatant to a new EP tube and place it on ice; (5) The remaining steps are basically the same as above, and the specific primary antibody preparation concentrations are as follows: mouse LCN2 antibody (1:1000); GAPDH-HRP antibody (1:5000); mouse Fibronectin antibody (1:1000); mouse Colleagen I antibody (1:1000); mouse α-SMA antibody (1:5000).

[0059] Experimental results are as follows Figure 4 and Figure 5 As shown, compared with the normal mouse control group, LCN2 expression in the peritoneum was significantly increased in both models of peritoneal dysfunction mice. Its expression level was negatively correlated with the D / D0 glucose ratio and positively correlated with D / Pur (the ratio of dialysate urea nitrogen to blood urea nitrogen), peritoneal thickness, and number of blood vessels.

[0060] The above results indicate that elevated LCN2 expression levels are associated with peritoneal dysfunction and can be used as a biomarker for early identification of peritoneal dysfunction in peritoneal dialysis patients.

[0061] Example 2 This embodiment analyzes the effect of LCN2 on peritoneal dysfunction.

[0062] Build Lcn2 Gene knockout mice ( Lcn2 - / - The study investigated the impact of LCN2 deficiency on peritoneal dysfunction. The specific experimental procedure included: SPF-grade male Lcn2 - / - C57BL / 6 mice were purchased from Guangdong Yaokang Biotechnology Co., Ltd., and housed at the Experimental Animal Center of the First Affiliated Hospital of Sun Yat-sen University during the modeling period. The construction of the acquired peritoneal dysfunction model mouse and the processing of animal specimens are described in Example 1.

[0063] AAV2-sh Lcn2 Treatment group: administered via intraperitoneal injection of sh Lcn2 (interference Lcn2Adeno-associated virus 2 (AAV2-shRNA) expressing gene Lcn2 ), to achieve the effect of peritoneal tissue Lcn2 Local knockdown of the gene was performed to assess the therapeutic potential of intervening in LCN2 for peritoneal dysfunction during disease progression. The specific experimental procedure included: SPF-grade male C57BL / 6 mice were purchased from Guangdong Yaokang Biotechnology Co., Ltd., and housed at the Experimental Animal Center of the First Affiliated Hospital of Sun Yat-sen University during the modeling period. 150 μL of AAV2-sh was injected intraperitoneally. Lcn2 The construction of the acquired peritoneal dysfunction model mouse and the processing of animal specimens are described in Example 1.

[0064] Using 5 Lcn2 - / - Mice, 5 AAV2-sh Lcn2 The treatment group mice and 5 wild-type mice (WT) were used to establish a mouse model of peritoneal dysfunction by intraperitoneal injection of glucose chlorhexidine (CG) and 4.25% glucose dialysate + LPS (PDS), respectively, with an equal volume of saline injected as a control.

[0065] The relevant testing procedures include: mouse PET test, HE staining, Masson staining, immunohistochemical staining of paraffin sections of mouse parietal peritoneum, qPCR and WB of mouse visceral peritoneum. For specific experimental steps, please refer to Example 1.

[0066] The results are as follows Figures 6A-6I As shown, in the two peritoneal dysfunction models constructed in different ways, compared with wild-type mice (WT), Lcn2 - / - The mice (KO) all showed significant remission of the pathological phenotype. Specifically, Masson and HE staining showed reduced peritoneal thickness; inflammatory factors ( Il6 , Il1b and Tnf The expression levels of AAV2-sh were significantly decreased, as were the infiltration of inflammatory cells (macrophages); CD31 and VEGFA-labeled angiogenesis were inhibited; and the expression of fibrosis markers (Fibronectin, α-SMA, Collagen I) was reduced. Similarly, compared to wild-type mice, AAV2-sh... Lcn2 The treated mice also exhibited similar characteristics after modeling. Lcn2 - / - Similar pathological improvements were observed in mice, including significant reductions in peritoneal thickness, inflammatory infiltration, angiogenesis, and fibrosis. Figures 7A-7H ).

[0067] The results above indicate that knocking out [the virus] in the peritoneal dysfunction model is effective. Lcn2Both gene knockdown and knockdown of LCN2 can effectively alleviate peritoneal dysfunction-related pathological phenotypes, indicating that LCN2 can serve as a potential therapeutic target for peritoneal dysfunction.

[0068] In summary, this invention provides an in-depth analysis of the mechanisms related to peritoneal dysfunction in peritoneal dialysis patients, identifies biomarkers for diagnosis and treatment, detects changes in LCN2 expression in peritoneal tissue and dialysis fluid of peritoneal dialysis patients and mouse models of peritoneal dysfunction, and analyzes its correlation with clinical indicators of peritoneal function, clarifying the relationship between LCN2 changes and acquired peritoneal dysfunction. An in vitro model of inflammatory fibrotic peritoneal mesothelial cells was constructed, and changes in LCN2 expression and its functional role in inflammatory fibrosis were observed. Furthermore, based on a mouse model of peritoneal dysfunction, a... Lcn2 Gene knockout mice, and mice carrying sh via intraperitoneal injection. Lcn2 Adeno-associated virus 2-mediated peritoneal localization Lcn2 Decreased gene expression levels were observed to investigate the role of LCN2 in regulating key pathological changes such as peritoneal inflammation, angiogenesis, and fibrosis. Knockout of LCN2 in a peritoneal dysfunction model was found to contribute to this effect. Lcn2 Both gene knockdown and knockdown of its expression level can effectively alleviate peritoneal dysfunction-related pathological phenotypes, indicating that LCN2 can be used to identify peritoneal dysfunction in peritoneal dialysis patients and as a target for developing drugs to treat peritoneal dialysis-related peritoneal dysfunction.

[0069] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A biomarker associated with peritoneal dysfunction, characterized in that, The biomarkers include lipid transporter 2.

2. Application of lipid transporter 2 and / or its detection reagents in the preparation of products for detecting peritoneal dysfunction.

3. The application according to claim 2, characterized in that, The detection reagents include reagents for detecting the expression level of lipid transporter protein 2.

4. A kit for detecting peritoneal dysfunction, characterized in that, The kit includes reagents for detecting the expression level of lipid transporter 2.

5. Application of lipid transporter 2 and / or its detection reagents in screening interventions for peritoneal dysfunction.

6. A method for screening interventions for peritoneal dysfunction, characterized in that, The method includes: administering a candidate intervention to an individual with peritoneal dysfunction and comparing the changes in lipid transporter 2 expression levels between the candidate intervention-treated group and the untreated control group; when the candidate intervention reduces the expression level of lipid transporter 2, the candidate intervention is deemed to have the potential to intervene in peritoneal dysfunction.

7. The method for screening interventions for peritoneal dysfunction according to claim 6, characterized in that, The candidate interventions include gene-editing agents for lipid transporter 2.

8. The method for screening interventions for peritoneal dysfunction according to claim 7, characterized in that, The gene-editing reagents for lipid transporter 2 include reagents that knock out the gene encoding lipid transporter 2 or knock down its expression level.

9. The application of gene-editing reagents for lipid transporter protein 2 in the preparation of interventions for peritoneal dysfunction, characterized in that, The gene editing reagents include reagents that knock out the gene encoding lipid transporter 2 or knock down its expression level.

10. An intervention for peritoneal dysfunction, characterized in that, The peritoneal dysfunction interventions include agents that knock out the gene encoding lipid transporter 2 or knock down its expression level.

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