Application of PCSK6 in the diagnosis of sepsis

Abstract

The present invention belongs to the field of biomedical technologies, and specifically relates to the use of a biomarker in the preparation of a product for diagnosing sepsis. The biomarker is proprotein convertase subtilisin / kexin type 6 (PCSK6), which has significantly high sensitivity and specificity in diagnosing sepsis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of PCSK6 in the preparation of products for diagnosing sepsis. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome caused by infection. Bacteria, viruses, and parasites can all trigger infection in the body. After the body is infected, components such as bacteria and toxins enter the body, activate the body's inflammatory response cells, and produce and release a large number of inflammatory mediators (such as TNF-α, IL-1β, IL-6, etc.), causing local or systemic inflammatory responses. In the state of severe sepsis, severe infections caused by immunosuppression will further exacerbate the systemic inflammatory response, and various pathological changes driven by the inflammatory response will further deteriorate, leading to or aggravating organ failure, and even death of the patient. Sepsis not only endangers human health but also brings a huge economic burden to medical and health care.

[0003] Currently, the diagnosis of sepsis mainly relies on the detection of blood, body fluids, or tissue specimens. Bacterial culture of the samples is used to determine the source of infection, and sometimes imaging examinations such as ultrasound examination, computed tomography (CT), and magnetic resonance imaging (MRI) are also used in combination to determine the source of infection. The time required for bacterial culture of the samples is relatively long, usually 1 - 3 days. Therefore, early diagnosis of sepsis is crucial. Early prevention and treatment can avoid the huge risks brought by sepsis to patients.

[0004] PCSK6 is a member of the proprotein convertase subtilisin / kexin (PCSKs) family, and the PCSKs family is also known as the serine protease family, including proteins with different functions, such as PCSK1 - 6, MBTPS1, etc.

[0005] PCSK1 participates in the processing of hormones and other protein precursors at sites composed of paired basic amino acid residues. Substrates include proopiomelanocortin (POMC), renin, enkephalin, dynorphin, somatostatin, insulin, and AGRP. PCSK1 activity is crucial for the activation cleavage of many peptide hormone precursors, which are related to the regulation of food intake, glucose homeostasis, and energy homeostasis (see Patent CN115698336A, publication date: November 11, 2021).

[0006] PCSK2 protease is packaged into dense core secretory granules and activated therein, and is expressed in the neuroendocrine system and the brain. Single nucleotide polymorphisms in this gene may increase the susceptibility to myocardial infarction and type 2 diabetes. This gene may also play a role in tumorigenesis and progression.

[0007] PCSK3 can process latent precursor proteins into their bioactive products. It is a calcium-dependent serine endoprotease that cleaves precursor proteins at their paired basic amino acid processing sites. Some furin substrates are: parathyroid hormone, transforming growth factor β1 precursor, proalbumin, pro-β-secretase, membrane type 1 matrix metalloproteinase, the β subunit of the nerve growth factor precursor, and von Willebrand factor.

[0008] PCSK4 is present in the acrosome of the sperm plasma membrane and plays an important role in the binding to zona pellucida (ZP) molecules during fertilization (see Patent IDP000069516B, publication date 2018-05-18). PCSK5 plays a key regulatory role in the secretion and maturation of growth factors.

[0009] PCSK6 is mainly involved in the processing of protein and peptide precursors and regulates protein trafficking and processing through the secretory pathway. It is expressed in many tissues, including neuroendocrine, liver, intestine, and brain, and plays a role in tumor progression. It has also been reported that the mRNA and protein levels of PCSK6 increase in hypoxic cardiomyocytes, thus identifying PCSK6 as a key participant in cardiac remodeling after acute myocardial infarction (see Non-Patent Literature: Secretome Analysis of Cardiomyocytes Identifies PCSK6 (Proprotein Convertase Subtilisin / Kexin Type 6) as a Novel Player in Cardiac Remodeling After Myocardial Infarction. Circulation. 2020;141:1628–1644.). In addition, it has been reported that PCSK6 may be a new antigenic target associated with non-steroidal anti-inflammatory drug use in membranous nephropathy (see Non-Patent Literature: Proprotein convertase subtilisin / kexin type 6 (PCSK6) is a likely antigenic target in membranous nephropathy and non-steroidal anti-inflammatory drug use. Kidney International (2023) 104, 343–352).

[0010] MBTPS1 encodes a membrane-bound enzyme called site-1 protease (SIP), which is a member of the serine protease family and acts as a proprotein convertase responsible for cleaving a variety of precursor substrates. Summary of the Invention

[0011] To fill the gap in the existing technology, the present invention uses PCSK6 as a biomarker for diagnosing sepsis, achieving high sensitivity and specificity. Specifically, in this application, a biomarker for sepsis - PCSK6 was screened out through a discovery cohort, and its diagnostic efficacy was further verified through a validation cohort.

[0012] In a first aspect of the present invention, there is provided an application of a biomarker or a reagent for detecting a biomarker in the preparation of a product for diagnosing sepsis, wherein the biomarker is proprotein convertase subtilisin / kexin type 6 (PCSK6).

[0013] The biomarker is a biomarker in cells, tissues or body fluids.

[0014] Preferably, the cells can be liver cells, brain cells or intestinal cells.

[0015] Preferably, the tissues can be liver tissues, brain tissues or intestinal tissues.

[0016] Preferably, the body fluids can be blood, plasma, serum, saliva, urine, tears, amniotic fluid, cerebrospinal fluid or lymph fluid.

[0017] The sepsis includes liver injury, brain injury, intestinal injury, gastric injury, lung injury, kidney injury or heart injury caused by sepsis.

[0018] Preferably it is sepsis-induced liver injury.

[0019] The product includes a kit, a test strip, a chip or a device.

[0020] The product includes a reagent for detecting the biomarker. Preferably, the reagent detects the presence or absence, protein expression level or concentration of the biomarker.

[0021] Preferably, the reagent for detecting the biomarker includes a reagent used by one or more of the methods of western blotting, enzyme-linked immunosorbent assay, chromatography or mass spectrometry.

[0022] The diagnosing of sepsis includes detecting the presence or absence, protein expression level or concentration of the biomarker. Preferably, it further includes comparing it with a threshold value, and the threshold value is obtained from previous experiments, that is, the threshold value is determined by analyzing the difference degree of the biomarker between sepsis patients and healthy people through experiments and data analysis.

[0023] In a specific embodiment of the present invention, the PCSK6 expression level or concentration has a difference or a significant difference from the threshold value (the difference is statistically significant, for example, log2(FoldChange)|(fold change)>0, P<0.05). For example: if PCSK6 is higher than the threshold value or significantly higher than the threshold value, it indicates the occurrence of sepsis, and the disease is diagnosed.

[0024] Preferably, the protein immunoblotting method, enzyme-linked immunosorbent assay, chromatography or mass spectrometry is used alone or in combination of two or more for detecting the biomarker.

[0025] More preferably, the enzyme-linked immunosorbent assay includes one or more of direct ELISA, indirect ELISA, double antibody sandwich ELISA or competitive ELISA.

[0026] More preferably, the chromatography includes one or more of gas chromatography, liquid chromatography, capillary electrophoresis, high performance liquid chromatography or ultra-high performance liquid chromatography.

[0027] More preferably, the mass spectrometry includes one or more of electrospray ionization mass spectrometry, quadrupole mass spectrometer, ion trap mass spectrometer, matrix-assisted laser desorption / ionization time-of-flight mass spectrometer, matrix-assisted laser desorption ionization-time of flight mass spectrometry, MALDI quadrupole-time of flight mass spectrometry, electrospray ionization (ESI)-TOF mass spectrometry, ESI ion trap mass spectrometry, ESI triple quadrupole mass spectrometry or Fourier transform ion cyclotron resonance.

[0028] In the second aspect of the present invention, there is provided an application of a biomarker in the preparation of a product for diagnosing sepsis liver injury, and the biomarker is proprotein convertase subtilisin / kexin type 6 (PCSK6).

[0029] In the third aspect of the present invention, there is provided an application of a biomarker in the construction of a diagnostic model for sepsis, and the biomarker is PCSK6.

[0030] In the fourth aspect of the present invention, there is provided a method for constructing a sepsis diagnostic model, and the construction method includes:

[0031] i): Collect the detection results of the biomarker expression level or concentration of sepsis patients and healthy controls, and the biomarker includes PCSK6;

[0032] ii): Use the results collected in i) to construct a diagnostic model; or,

[0033] The construction method includes:

[0034] 1) Collect samples from the subject and detect the expression level or concentration of the biomarker, where the biomarker includes PCSK6;

[0035] 2) Clinically diagnose the subject and divide the subject into a sepsis patient group and a healthy control group;

[0036] 3) Construct a diagnostic model based on the detection result of step 1) and the diagnosis result of step 2).

[0037] In the fifth aspect of the present invention, there is provided a diagnostic model obtained by the above construction method.

[0038] In the sixth aspect of the present invention, there is provided a method for diagnosing sepsis, the method including detecting the presence, expression level or concentration of PCSK6 in a sample of the subject.

[0039] The sample includes cells, tissues or body fluids.

[0040] Preferably, the cells can be liver cells, brain cells or intestinal cells.

[0041] Preferably, the tissues can be liver tissues, brain tissues or intestinal tissues.

[0042] Preferably, the body fluids can be blood, plasma, serum, saliva, urine, tears, amniotic fluid, cerebrospinal fluid or lymph fluid.

[0043] The method includes one or more of Western blot, enzyme-linked immunosorbent assay, chromatography or mass spectrometry.

[0044] Preferably, the method includes comparing the detected expression level or concentration of the biomarker with a threshold value, where the threshold value is obtained from previous experiments, that is, the threshold value is determined by the degree of difference in the biomarker between sepsis patients and healthy people through experiments and data analysis. The biomarker described in this application has a difference or a significant difference from the threshold value (the difference is statistically significant, for example, log2(FoldChange)|(fold change)>0, P<0.05). For example: PCSK6 being higher than the threshold value or significantly higher than the threshold value indicates the occurrence of sepsis, and the disease is then diagnosed.

[0045] The term "subject" can be a human or a non-human animal, including "patients", "suspected patients" and "healthy individuals", etc. This term does not indicate a specific age or gender, covering adult or neonatal subjects as well as fetuses. The non-human animal can be a wild animal, a zoo animal, an economic animal, a pet, a laboratory animal, etc. Specifically, the non-human animal includes but is not limited to pigs, cows, sheep, horses, donkeys, foxes, raccoons, minks, camels, dogs, cats, rabbits, rats (such as rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels) or monkeys, etc.

[0046] The term "diagnosis" refers to ascertaining whether a patient has a disease or disorder in the past, at the time of diagnosis, or in the future, or to ascertaining the progression or possible future progression of a disease. Brief Description of the Drawings

[0047] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:

[0048] Figure 1 Shown is a Venn diagram of differential protein analysis in peripheral serum of sepsis patients and normal control groups;

[0049] Figure 2 Shown is a sample correlation heat map;

[0050] Figure 3 Shown is a principal component analysis diagram (PCA analysis);

[0051] Figure 4 Shown is a bar chart drawn based on the total number of differential proteins and the specific number of up-regulated / down-regulated proteins, where up represents up-regulation and down represents down-regulation;

[0052] Figure 5 Shown is a volcano plot of differential proteins, where up represents up-regulation, down represents down-regulation, and nosig represents no significant difference;

[0053] Figure 6 Shown is a GO (Gene Ontology Enrichment Analysis) enrichment map;

[0054] Figure 7 Shown is a protein expression level diagram of PCSK6 in sepsis patients and healthy individuals, **** represents p<0.0001;

[0055] Figure 8 Shown is a receiver operating characteristic curve (ROC), where 95% CI is 0.6738 - 1.000, and CI is the confidence interval;

[0056] Figure 9 Shown is a bar chart of the change in liver function index concentration over time, where Figure A is alanine aminotransferase (ALT), Figure B is aspartate aminotransferase (AST), Figure C is total bilirubin (TBIL), Figure D is γ-glutamyl transpeptidase (γ-GT), Figure E is albumin (ALB), Figure F is alkaline phosphatase (ALP), Figure G is total bile acid (TBA), CLP represents the sepsis model group, SHAM represents the sham operation model group, ns represents no significant difference, * represents p<0.05, ** represents p<0.01, *** represents p<0.001, **** represents p<0.0001;

[0057] Figure 10 The figure shows the structural diagram of HE staining (hematoxylin - eosin staining). CLP represents the sepsis model group, and SHAM represents the sham - operation model group;

[0058] Figure 11 The figure shows the results of pathological scores of liver injury at different times. CLP represents the sepsis model group, SHAM represents the sham - operation model group, * represents p < 0.01, and **** represents p < 0.0001;

[0059] Figure 12 The figure shows the relative protein expression of PCSK6 in hepatocytes of sepsis - model mice and sham - operation - model mice. * represents p < 0.01, CLP represents the sepsis model group, and SHAM represents the sham - operation model group;

[0060] Figure 13 The figure shows the protein concentration of PCSK6 in the serum of sepsis - model mice and sham - operation - model mice. **** represents p < 0.0001, CLP represents the sepsis model group, and SHAM represents the sham - operation model group;

[0061] Figure 14 The figure shows the receiver operating characteristic curve (ROC) of the model mice. Among them, 95% CI is 0.9420 - 1.000, and CI is the confidence interval. Detailed implementation manners

[0062] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only partial embodiments of the present invention, rather than all. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0063] It should be noted that the methods used in the present invention are all conventional methods unless otherwise specified, and the reagents used in the present invention are all commercially available products unless otherwise specified.

[0064] Animals used in the examples: C57BL / 6J mice, weighing about 20 g, male, healthy specific - pathogen - free grade, Beijing Huafukang Biotechnology Co., Ltd., license number: SCXK(Beijing)2019 - 0008. They were fed with a full - price nutritional diet, at room temperature of 22 - 25 °C, and humidity of 30% - 50%.

[0065] Instruments used in the examples: DYY-7 protein electrophoresis instrument (Beijing Liuyi Instrument Factory), ImageQuant LAS4000 chemiluminescence imaging analyzer (GE General Electric Company), Spectra MR multifunctional microplate reader (DYNEX Technologies Company), Vanquish Neo chromatograph (Thermo Company), Astral liquid chromatography tandem mass spectrometer (Thermo Company).

[0066] All data in the examples of this application are expressed as x±s. The comparison between groups is a one-way analysis of variance. P<0.05 indicates a statistically significant difference, and the SPSS 25.0 software package is used for statistical processing.

[0067] Example 1 Screening of biomarkers for diagnosing sepsis

[0068] 1. Discovery cohort

[0069] Participants were recruited, and whole blood samples were collected from the Chinese PLA General Hospital. The diagnosis of the disease population was based on the Sepsis3.0 definition and SOFA score. The exclusion criteria were malignant tumors and chronic respiratory diseases, excluding any confounding factors of anticoagulant drugs. The normal population had normal physical examinations and did not have the above diseases. Based on the above diagnostic criteria, the discovery cohort consisted of 11 confirmed sepsis patients and 10 healthy controls.

[0070] 2. Detection of proteins in samples

[0071] The human whole blood samples were allowed to stand to obtain serum. Filter columns were used to remove high-abundance proteins, and the protein concentration was measured by the BCA method. Subsequently, the samples were subjected to protein denaturation, reduction, alkylation, and enzymatic digestion, and the Waters-C18 SPE column was used for sample desalting, followed by elution, concentration, reconstitution, and the supernatant was taken for on-machine detection for Astral-DIA proteomics analysis.

[0072] 3. Results

[0073] A total of 4910 proteins were detected by Astral-DIA proteomics analysis, of which 4794 proteins were common to the sepsis patient group and the healthy control group ( Figure 1 ). There were significant differences between the two groups of samples, and there was a strong correlation between the samples within each group, and the data reliability was high ( Figure 2 、 Figure 3 ). According to the set threshold |log2(FoldChange)| (fold change)>0, P<0.05, cluster analysis was performed on the differentially expressed proteins between the groups ( Figure 4 、 Figure 5), 20 differential proteins with significantly different serum protein expression levels in sepsis patients compared to the healthy control group were screened out, including CD14, PRH1, WFDC2, PGLYRP1, REG1B, RETN, GXYLT1, CELA2A, ATG, MYL3, PCSK6, etc. Further, through GO enrichment pathway analysis ( Figure 6 ), PCSK6 was selected as a diagnostic marker for sepsis, and its expression was significantly upregulated in sepsis patients ( Figure 7 ), with a significant difference.

[0074] Example 2 Verification of PCSK6 in the Diagnosis of Sepsis

[0075] To further verify the diagnostic efficacy of PCSK6 in sepsis, in this example, 20 new sepsis patients and 10 healthy controls were newly collected from the Chinese PLA General Hospital to form a verification cohort. The patient inclusion and exclusion criteria were consistent with those in the discovery cohort in Example 1 for omics screening, and whole blood samples of sepsis patients and normal populations were collected.

[0076] Subsequently, serum in the whole blood samples was taken for ELISA determination. The results showed that PCSK6 was highly specifically expressed in the serum of sepsis patients compared to the healthy control, and it was used to draw the Receiver Operating Characteristic (ROC) curve, as shown in Figure 8 , where AUC = 0.85, the cut-off value was 0.75, the sensitivity was 95%, and the specificity was 80%.

[0077] Example 3 Expression of PCSK6 in a Mouse Sepsis Model

[0078] 1. Experimental Design

[0079] Twenty C57BL / 6J male mice were divided into 2 groups according to the random number table method, with 10 mice in each group. One group was used to prepare a mouse sepsis model by cecal ligation and puncture. After the mice were routinely anesthetized, the abdomen was opened, the cecum was found and bluntly dissected. Ligation was performed at a distance of 1.0 - 1.5 cm from the distal end of the cecum, and then the cecum was punctured through with a needle, and feces the size of sesame seeds were extruded from the puncture hole. The cecum was then returned to the abdominal cavity, and the abdominal wall muscles and skin were sutured layer by layer. The other group was used to prepare a mouse sham operation model by the sham operation method. After the mice were routinely anesthetized, the abdomen was opened, and the cecum was bluntly dissected to avoid bleeding and damage to the mesenteric blood vessels. Then the abdominal wall muscles and skin were sutured layer by layer.

[0080] 2. Protein Expression Detection

[0081] 1) Detection of PCSK6 protein expression in mouse hepatocytes

[0082] At 24 h after surgery, two groups of mice were anesthetized by intraperitoneal injection of 3% sodium pentobarbital at a dose of 0.2 ml / 100 g, and then liver perfusion was performed. After perfusion, the livers were removed and primary hepatocytes were isolated and extracted by density centrifugation. Approximately 2×10 6 cells were collected from each group. After extracting the total cellular proteins, the protein expression of PCSK6 in the cells was detected by Western blotting. The primary antibodies were rabbit anti-mouse PCSK6 polyclonal antibody and mouse anti-mouse β-actin monoclonal antibody (both diluted at 1:1000); the secondary antibodies were HRP (horseradish peroxidase)-labeled goat anti-rabbit IgG monoclonal antibody and HRP-labeled goat anti-mouse IgG monoclonal antibody (both diluted at 1:5000). A hypersensitive chemiluminescence kit was used for development and imaging. After observation with a chemiluminescence imaging analyzer, the gray value of the target protein was analyzed using ImageJ software (National Institutes of Health, USA). β-actin was used as an internal reference, and the relative expression level of the target protein was calculated.

[0083] 2) Detection of PCSK6 protein expression in the peripheral serum of mice

[0084] At 24 h after surgery, whole blood samples were collected from two groups of mice by orbital enucleation. Then, the whole blood was placed in a centrifuge and centrifuged at 3000 r / min for 10 minutes to obtain serum. The PCSK6 level in the obtained serum was detected using an ELISA kit provided by Shanghai Aimeng Youning Biotechnology Co., Ltd. The ELISA experiment was performed and calculated strictly according to the kit operation instructions to minimize human error.

[0085] 3. Liver injury identification

[0086] The liver function indexes ALT, AST, TBIL, γ-GT, ALP, ALB, and TBA of each group of mice were detected at different time points, and the liver injury was evaluated by combining HE staining and liver injury pathological scores.

[0087] 4. Experimental results

[0088] 1) Liver injury in the sepsis model

[0089] The levels of ALT, AST, TBIL, γ-GT, ALP, and TBA in the liver tissues of mice showed a trend of first increasing and then decreasing in a time-dependent manner, with the most obvious increase at 12 h and 24 h after CLP surgery and a decrease at 48 h; the level of ALB was the lowest at 24 h after CLP and recovered at 48 h ( Figure 9 ). HE staining ( Figure 10 ) and liver injury pathological scores ( Figure 11)Results showed that compared with the Sham group, the liver tissues of mice in the 6 h, 12 h, and 24 h groups after CLP showed varying degrees of injury, which progressively worsened over time. The main manifestations were disordered hepatic cord structure, inflammatory cell infiltration, and hepatocyte edema and degeneration. However, the above pathological injuries in the liver tissue of the 48 h group after CLP modeling were significantly alleviated compared with those in the CLP-24 h group. This, together with the liver function indicators, suggested that under septic conditions, liver tissue injury increased in a time-dependent manner and was most significant at 24 h of sepsis and decreased at 48 h.

[0090] 2) PCSK6 protein was highly specifically expressed in the septic mouse model. Specifically:

[0091] Hepatocytes: Twenty-four hours after cecal ligation and puncture in mice, primary hepatocytes were isolated and extracted, and total cellular proteins were extracted for Western blotting. The results were as Figure 12 shown. Compared with the sham operation model group, the expression of PCSK6 in hepatocytes of the septic model group mice was significantly increased, and the difference was significant.

[0092] Serum: Twenty-four hours after cecal ligation and puncture in mice, blood was collected from the eye socket, and serum was separated. The expression level of PCSK6 in the serum of mice was detected using an ELISA kit. The results were as Figure 13 shown. Compared with the sham operation model group, the level of PCSK6 in the peripheral serum of the septic model group mice was significantly increased, and the difference was significant.

[0093] Example 4: Diagnostic verification in the mouse sepsis model

[0094] Another 40 mice were randomly divided into groups by the random number method. Twenty mice were randomly selected as the control group for sham operation, and the other 20 mice were used as the CLP group for cecal ligation and puncture (the preparation methods of the sham operation model and the CLP model were referred to the methods in Example 3). Twenty-four hours after the operation, hepatocytes were isolated and extracted respectively, thoroughly ground and lysed, and then centrifuged for 5 minutes. The supernatant was taken for ELISA experiments to detect the content of PCSK6 protein in the liver tissue, and an ROC curve was formed to predict the diagnostic value.

[0095] Liver injury occurred in the constructed CLP model, which was consistent with the results of Example 3. The expression level of PCSK6 protein in hepatocytes of the CLP model group was significantly higher than that in the sham operation group, and the ROC results were as Figure 14 shown, with AUC = 0.975, the cut-off value was 0.85, the sensitivity was 100%, and the specificity was 85%.

Claims

1. Use of a biomarker in the preparation of a product for diagnosing sepsis, characterized in that, The biomarker is proprotein convertase subtilisin / kexin type 6.

2. The application according to claim 1, wherein The biomarker is a biomarker in blood, plasma, or serum.

3. The application according to claim 1 or 2, characterized in that, The sepsis includes liver injury, brain injury, intestinal injury, gastric injury, lung injury, kidney injury, or heart injury caused by sepsis.

4. The application according to claim 1, characterized in that The product includes a kit, test strip, chip, or device.

5. The application according to claim 1, wherein The product includes a reagent for detecting the biomarker, and the reagent detects the presence or absence, protein expression level, or concentration of the biomarker.

6. The application according to claim 1, wherein The diagnosis of sepsis includes detecting the presence or absence, protein expression level, or concentration of the biomarker.

7. The application according to claim 5 or 6, characterized in that, The detection of the biomarker uses one or more of Western blot, enzyme-linked immunosorbent assay, chromatography, or mass spectrometry.

8. Use of a biomarker in the preparation of a product for diagnosing sepsis-induced liver injury, characterized in that, The biomarker is proprotein convertase subtilisin / kexin type 6.

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