Ligand of platelet activating factor receptor and application of ligand in preparation of health monitoring preparation

Abstract

The invention provides a ligand of a platelet activating factor receptor (PTAFR) and application of the ligand in preparation of a health monitoring preparation. The invention verifies that PE 18: 0 / 18: 1 is increased along with the decrease of renal function, verifies the existence of Ptafr endogenous ligand at a clinical level, explores the potential of the Ptafr endogenous ligand as a novel biomarker for clinical AKI patients (ATN), and can effectively distinguish healthy people (HLD) from AKI patients.

Description

Technical Field

[0001] The present invention belongs to the field of chemical medicine technology, and specifically relates to the pharmaceutical use of PE18:0 / 18:1 as a ligand, and especially to a ligand of platelet-activating factor receptor and its application in the preparation of health monitoring preparations. Background Art

[0002] The platelet-activating factor receptor (PTAFR) is a member of the G protein-coupled receptor A family with seven transmembrane spans. Its extracellular N-terminus is short, and its transmembrane region readily forms a ligand-binding pocket, primarily functioning as a signal transducer in vivo. When PTAFR binds to platelet-activating factor (PAF), it activates the downstream G protein Gαq, initiating a series of intracellular signaling pathways that regulate the body's inflammatory response. Recent studies have shown that PTAFR can be activated in tissues lacking its classic ligand, such as the renal tubules, suggesting the existence of other, unknown ligands for PTAFR. The discovery of novel ligands for PTAFR is also of great value in drug development.

[0003] Lipidomics is a research model based on high-throughput analytical technology that systematically analyzes changes in lipid composition and expression in organisms. Lipidomics analysis can efficiently study the changes and functions of lipid families and lipid molecules in various biological processes, and then elucidate the relevant biological activity processes and mechanisms. Lipidomics has shown broad application prospects in the identification of disease lipid biomarkers, disease diagnosis, discovery of drug targets and lead compounds, and research on drug action mechanisms. Lipidomics analysis generally uses liquid chromatography-mass spectrometry (LC-MS) technology, which is mainly divided into two categories: non-targeted analysis (untargeted) and targeted analysis (targeted). Among them, the non-targeted analysis mode can achieve unbiased systematic analysis of various types of lipids in the sample. Summary of the Invention

[0004] The present invention aims to screen and discover new ligands for PTAFR. Through non-targeted lipidomics, molecular docking, PLO and other experiments, this application discovered for the first time that PE18:0 / 18:1 can serve as a ligand for platelet-activating factor receptor (PTAFR), and then proposed a new biological function of PE18:0 / 18:1 in renal tubular cells, which can be used to distinguish healthy people (HLD) from patients with acute kidney injury (ATN).

[0005] The present invention provides a ligand of platelet-activating factor receptor (PTAFR), which comprises PE18:0 / 18:1.

[0006] The present invention further provides use of phosphatidylethanolamine as a ligand of platelet-activating factor receptor (PTAFR), wherein the phosphatidylethanolamine is PE18:0 / 18:1.

[0007] The present invention also provides the use of phosphatidylethanolamine in preparing a preparation for diagnosing renal function-related diseases, wherein the phosphatidylethanolamine is PE: 18:0 / 18:1.

[0008] In one embodiment of the present invention, the renal function-related disease is acute tubular necrosis (ATN).

[0009] The present invention verifies the existence of Ptafr endogenous ligand at the clinical level and explores its potential as a new biomarker for clinical AKI patients (ATN). The present invention verifies that PE 18:0 / 18:1 increases with the decline of renal function and can effectively distinguish between HLD and AKI patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 PTAFR signaling was examined in control and hypoxia-reoxygenation-treated mouse primary renal tubular cells for β-arrestin recruitment experiments. *** P < 0.001, ** P < 0.01, * P < 0.05; Figure 2 Figure 1 is a diagram of the results of non-targeted lipidomics testing, where A is a schematic diagram of non-targeted lipidomics sample collection; B is a diagram of lipid distribution in the supernatant of in vitro mouse primary renal tubular cell culture using non-targeted lipidomics testing; Figure 3 The figure shows the KEGG analysis results of differential lipids in the culture supernatants of primary renal tubular cells of control and hypoxia-reoxygenation treated mice; Figure 4 Figure 2 shows the results of targeted lipidomics testing. A shows the abundance of various glycerophospholipids in the culture supernatants of primary renal tubular cells from mice subjected to control and hypoxia-reoxygenation treatments; B shows the content of PE18:0 / 18:1 in the culture supernatants of primary renal tubular cells from mice subjected to control and hypoxia-reoxygenation treatments. *** P < 0.001, ** P < 0.01, * P < 0.05. Figure 5 Heatmap of the abundance of the top 15 lipids scored for VIP Figure 6 Schematic diagram of the binding of ligand PE (18:0 / 18:1) to receptor PTAFR, where A is the molecular docking showing the binding pocket of ligand PE (18:0 / 18:1) to receptor PTAFR; B is the protein lipid co-sedimentation experiment verifying the binding detection results of ligand PE (18:0 / 18:1) to receptor PTAFR; PLO, Protein Lipid Overlay assay; Figure 7Figure 1 shows the effect of PE (18:0 / 18:1) on HK2 cells. Figure A shows the effect of PE (18:0 / 18:1) on the cycle of human immortalized renal tubular HK2 cells by flow cytometry; Figure B shows the expression of CyclinB / CyclinD in HK2 cells after treatment with PE (18:0 / 18:1) and siPTAFR by Western Blot. siPTAFR: small interfering siRNA that silences the PTAFR receptor. Figure 8 Figure 1 is a diagram of lipidome analysis results, where A is a flowchart of urine sample grouping and collection for the targeted lipidome; Figure 1 is a diagram of the differential lipids in normal human urine and acute tubular injury urine using KEGG enrichment analysis; Figure 9 This is a schematic diagram of the distribution of various types of glycerophospholipids in the clinical urine lipid panel; Figure 10 Figure 2 shows the distribution of PE in clinical urine. (A) shows the distribution of glycerophosphatidylethanolamine (PE) by fatty acid chain subclass in the clinical urine lipid panel; (B) shows the abundance of PE 18:0 / 18:1 in the clinical urine lipid panel. ***, P < 0.001.

[0011] Figure 11 The purpose of this study was to analyze the ROC of the clinical urine lipid panel to determine the discrimination between PE 18:0 / 18:1 in the clinical urine lipid panel and normal subjects and patients with acute tubular necrosis. DETAILED DESCRIPTION

[0012] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0013] Unless otherwise specified, all reagents used in this example were of analytical grade, and the progress of all chemical reactions was monitored by thin-layer chromatography.

[0014] Example 1 Extraction of primary mouse renal tubular cells Mouse renal cortical tissue was dissected and minced, then transferred to PBS containing 0.1% type II collagenase (A004174, Sangon, Shanghai, China) and digested for 10 minutes at 37°C. Digestion was then terminated with DMEM / F12 medium (Gibco, Rockville, MD, USA) supplemented with 10% fetal bovine serum (FBS, Corning, New York, USA). The minced tissue was filtered through a 70 μm cell strainer (Beyotime, Shanghai, China) and centrifuged at 900 g for 4 minutes. The supernatant was removed, and the renal tubular tissue was resuspended in complete culture medium (CM) and incubated at 37°C in a 5% CO2 incubator until ready for use.

[0015] Example 2 β-arrestin-1 recruitment experiment

[0016] 293 cells were co-transfected with the PTAFR-eYFP and β-arrestin-1-RLUC plasmids and placed in a cell culture incubator. After 24 hours, the cells were replated in a 96-well plate and incubated at 37°C for 24 hours. The cells were washed twice with Tyrode's solution and ligand treatment was performed. The luciferase substrate coelenterazine-h (5 μM) was added and detected using a multi-microplate reader. The BRET signal was determined by calculating the luminescence ratio at 530 nm / 485 nm.

[0017] The results are as follows Figure 1 As shown in the figure, compared with the control group, the β-arrestin-1 recruitment experimental signal was stronger in the HR intervention group, which suggested that there were ligands that could activate PTAFR in the supernatant of primary renal tubular cells treated with HR.

[0018] Example 3 Lipidomics

[0019] A non-targeted lipidomics analysis platform based on UPLC-MS / MS (QE Plus™) liquid chromatography mass spectrometry system was used in combination with MSDAIL software for lipid identification and data preprocessing. Figure 2 As shown in Figure A, MSDAIL software integrates raw data processing, peak extraction, lipid identification, peak alignment, and quantification. Based on the high-resolution, high-quality data generated by an Orbitrap mass spectrometer, it utilizes algorithms for identifying product ions, precursor ions, and neutral loss scans to enable systematic and reliable qualitative lipid analysis.

[0020] A total of 1126 lipids were detected by lipidomics ( Figure 2 B). KEGG analysis of differential lipids showed that differential lipids were mainly enriched in the glycerophospholipid metabolism pathway ( Figure 3 ).

[0021] Samples were separated using a Nexera X2 LC-30AD ultrahigh-pressure liquid chromatography system (Shimadzu). Lipid mass spectrometry analysis was performed using a QTRAP 5500 mass spectrometer (AB SCIEX) in positive and negative ion modes. Multiple reaction monitoring (MRM) was used for targeted lipidomics analysis of target lipids.

[0022] We further extracted the culture supernatants of primary renal tubular cells from the control and HR groups of mice and performed targeted lipidomics, focusing on glycerophospholipids. The results showed that compared with the control group, the abundance of LPC lipids in the HR group decreased most significantly, while the abundance of PE, PC, and PI lipids increased ( Figure 4 ) Heatmap and VIP scoring methods: 1. Logarithmic transformation and UV formatting were performed on the data. OPLS-DA modeling and analysis were first performed on the first principal component. The quality of the model was tested using 7-fold cross validation. The cross-validated R2Y (the degree to which the model explains the categorical variable Y) and Q2 (the predictive power of the model) were then used to assess the model's effectiveness. Finally, a permutation test was performed to randomly change the order of the categorical variable Y 200 times to obtain different random Q2 values, thereby further testing the model's effectiveness.

[0023] 2. Calculate the variable importance for the projection (VIP) to measure the influence and explanatory power of each metabolite expression pattern on the classification and discrimination of each group of samples, thereby assisting in the screening of marker metabolites The heat map shows the abundance of the top 15 lipids in VIP score ( Figure 5 ).

[0024] Example 4 Molecular Docking

[0025] The crystal structure of the PTAFR_HUMAN protein (PDB ID: 5ZKP) was downloaded from the PDB database. The receptor molecule was hydrogenated and charged. The 2D / 3D structures of each lipid group were downloaded from the HMD database (SDF files). Molecular docking of the lipids and receptor was performed using Autodock vina with the SR 27417 binding site as the center point. The results are shown in Figure 2. Figure 6 As shown in A.

[0026] Example 5 Protein Lipid Overlay assay (PLO)

[0027] PVDF membranes were preincubated with target lipids (Sigma, USA) for 1 hour. The membranes were then blocked with 5% skim milk (Millipore, USA) for 1 hour, washed with TBS, and gently incubated with a solution containing 1–5 μg / ml PTAFR protein for 1.5 hours at room temperature. The membranes were then washed with TBS and incubated with a PTAFR primary antibody for 1 hour at room temperature. The membranes were then washed with TBS and incubated with the corresponding secondary antibody for 1 hour at room temperature. Lipid-bound proteins bound to the membranes were detected by enhanced chemiluminescence (ECL).

[0028] The results are as follows Figure 6 As shown in B.

[0029] Molecular docking and PLO verified the binding of PE (18:0 / 18:1) to PTAFR at the computer and biological levels ( Figure 6 ). Example 6 Flow cytometry cell cycle detection

[0030] Cells for cell cycle analysis were collected and fixed with 70% ethanol overnight at 4°C. The cells were then stained in phosphate-buffered saline containing propidium iodide (PI) (10 mg / ml) and RNase A (50 mg / ml) and analyzed by flow cytometry (Beckman Coulter, USA).

[0031] The effect of PE (18:0 / 18:1) on the cell cycle of human immortalized renal tubular HK2 cells was detected by flow cytometry. The results showed that PE (18:0 / 18:1) treatment can lead to G2 / M phase arrest of HK2 cells. Knockdown of PTAFR by siRNA can improve the G2 / M phase arrest. The ratio of cyclin B / Cyclin D also obtained consistent results ( Figure 7 ).

[0032] Methods for effectively identifying patients with AKI 1. Twenty patients aged 18 to 75 years diagnosed with AKI and 18 healthy controls were enrolled. Patients with inflammatory and autoimmune diseases, liver dysfunction, polycystic kidney disease, diabetes, and pregnancy were excluded. Urine was collected from the subjects, centrifuged, and the supernatant was submitted for targeted lipidomics analysis.

[0033] 2. Samples were separated using a Nexera X2 LC-30AD ultra-high pressure liquid chromatography system (Shimadzu). The mobile phase was as follows: Solvent A consisted of 50% acetonitrile and 10 mM ammonium acetate (pH 8.0), and Solvent B was 100% acetonitrile. The sample was reconstituted in 120 μL of a 1:1 dichloromethane-methanol solution and stored in an autosampler at 4°C. The column temperature was set at 40°C, the flow rate was 300 μL / min, and the injection volume was 3 μL. The gradient elution program was as follows: 0–0.1 min, 85% B; 0.1–7.5 min, linear decrease of B from 85% to 65%; 8.5–11 min, linear decrease of B from 65% to 5%; 11–11.1 min, linear increase of B from 5% to 85%; 11.1–15 min, 85% B. Quality control (QC) samples were inserted throughout the sample sequence to evaluate the stability and reproducibility of the system.

[0034] 3. Mass spectrometry analysis was performed using a QTRAP 5500 mass spectrometer (AB SCIEX) in positive and negative ion modes. The ESI source conditions on the QTRAP 5500 were as follows: Positive ion mode: source temperature 550°C, ion source gas 1 (GAS1): 40, ion source gas 2 (GAS2): 50, curtain gas (CUR): 35, ion spray voltage (ISVF) 5500 V. Negative ion mode: source temperature 550°C, ion source gas 1 (GAS1): 40, ion source gas 2 (GAS2): 50, curtain gas (CUR): 35, ion spray voltage (ISVF) -4500 V. Multiple reaction monitoring (MRM) mode was used to detect the target lipid pathway.

[0035] 4. After reading the data, cluster analysis was performed using methods such as PLS-DA and OPLS-DA, difference analysis was performed using methods such as variation fold analysis and T test, and enrichment analysis was performed using methods such as KEGG.

[0036] The existence of Ptafr endogenous ligand was verified at the clinical level, and its potential as a new biomarker for clinical AKI patients (ATN) was explored. Since the kidneys form urine through filtration and reabsorption, urine is often used as a carrier of renal biomarkers in kidney disease research. Therefore, the present invention collected urine from 18 healthy individuals (HLD) and 20 AKI patients for targeted lipidomics. KEGG analysis showed that the differential lipids were mainly enriched in glycerophospholipid metabolism ( Figure 8), which is consistent with the results of the above-mentioned mouse primary renal tubular cell supernatant. Among the lipids with different urine samples, PE was the most abundant, followed by PC and PG ( Figure 9 ).

[0037] Subgroup analysis of PE subtypes revealed that PE 18:0 / 18:1 was the most abundant ( Figure 10 Compared with the control group (Healthy living donor, HLD), the AKI group had higher urine PE 18:0 / 18:1 content ( Figure 10 ). The ROC curve analysis showed that PE 18:0 / 18:1 had a discriminative ability / differentiation for HLD and AKI, and the area under the ROC curve (AUC) was 0.928 ( Figure 11 These results suggest that PE 18:0 / 18:1 increases with declining renal function and can effectively distinguish patients with HLD from patients with AKI.

[0038] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

A ligand for platelet-activating factor receptor (PTAFR), comprising PE18:0 / 18:

1.

2. Use of phosphatidylethanolamine as a ligand for platelet-activating factor receptor (PTAFR), wherein The phosphatidylethanolamine is PE18:0 / 18:

1.

3. Use of phosphatidylethanolamine in the preparation of a preparation for monitoring renal function-related diseases, wherein: The phosphatidylethanolamine is PE:18:0 / 18:

1.

4. The use according to claim 3, characterized in that The renal function-related disease is acute tubular necrosis (ATN).

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