A vascular inflammation-related protein marker for assisting diagnosis or detection of cerebral aneurysms and its application

Abstract

The invention discloses a vascular inflammation-related protein marker for auxiliary diagnosis or detection of cerebral aneurysm, characterized in that the protein marker is a combination of OLR1 protein, IL6 protein, PTX3 protein and LPL protein or OLR1 protein, and the prepared kit for auxiliary diagnosis or detection of cerebral aneurysm comprises RT-qPCR quantitative amplification primers of OLR1 transcriptome, IL6 transcriptome and PTX3 transcriptome. The advantage is that the detection kit based on detecting the transcriptome / protein expression level of OLR1 marker, IL6, PTX3, OLR1, LPL combination marker and OLR1, IL6 and PTX3 combination marker can conveniently and quickly realize the detection of cerebral aneurysm at the molecular level, and has high detection efficiency, high sensitivity and strong pertinence.

Description

Technical Field

[0001] The present invention relates to the technical field of auxiliary diagnosis of cerebral aneurysms, and in particular to a vascular inflammation-related protein marker for auxiliary diagnosis or detection of cerebral aneurysms and an application thereof. Background Art

[0002] Brain aneurysm ( Intracranial aneurysms IA) is a potentially fatal asymptomatic disease with high morbidity and mortality, especially affecting people aged 40 to 70 years. Cerebral aneurysms are characterized by abnormal dilation of cerebral arteries, which may remain undetected for many years until symptoms occur through compression of intracranial nerves or catastrophic rupture leading to subarachnoid hemorrhage. Approximately 30-50% of patients die from the disease if they are not treated promptly after rupture of a cerebral aneurysm, and approximately 30% of patients who are treated in time after rupture of a cerebral aneurysm will be left with varying degrees of disability, and few survivors can return to normal life. Therefore, we urgently need a technology that can diagnose and predict cerebral aneurysms at an early stage.

[0003] The detection of protein biomarkers plays a vital role in disease prediction, screening, treatment, and long-term outcome evaluation. Data from animal model studies of cerebral aneurysms show that macrophages infiltrate the arterial wall at the onset of the disease. Subsequently, inflammatory factors such as tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) lead to elastic membrane loss and collagen matrix damage, promoting aneurysm formation through outward remodeling. Persistent inflammation is characterized by infiltration of macrophages, T lymphocytes, and B lymphocytes, which ultimately leads to tissue fibrosis, leading to rupture of cerebral aneurysms and life-threatening conditions. Therefore, free inflammation-related proteins in the blood have great potential as biomarkers for the diagnosis of early cerebral aneurysms.

[0004] Oxidized low-density lipoprotein receptor 1 (abbreviated as OLR1) is a pattern recognition receptor that plays an important role in cardiovascular and cerebrovascular diseases. In cerebrovascular diseases, OLR1 can promote oxidative stress and produce a large amount of oxidized low-density lipoprotein (abbreviated as ox-LDL). After OLR1 binds to ox-LDL, it activates a series of signaling pathways, further aggravates oxidative stress, promotes inflammatory response, and leads to vascular endothelial damage and vascular lesions. At present, there are few research reports on the relationship between OLR1 and cerebral aneurysms at home and abroad. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a vascular inflammation-related protein marker for auxiliary diagnosis or detection of cerebral aneurysms with high detection efficiency, high sensitivity and strong specificity and its application.

[0006] The technical solution adopted by the present invention to solve the above technical problem is: a vascular inflammation-related protein marker for auxiliary diagnosis or detection of cerebral aneurysms, wherein the protein marker is OLR1 protein.

[0007] Furthermore, the above-mentioned vascular inflammation-related protein marker is used in the preparation of a kit for auxiliary diagnosis or detection of cerebral aneurysm, wherein the kit includes RT-qPCR quantitative amplification primers for the OLR1 transcriptome, the nucleotide sequence of the forward amplification primer of the OLR1 transcriptome is shown in SEQ ID NO.1: 5′-TTGCCTGGGATTAGTAGTGACC-3′, and the nucleotide sequence of the reverse amplification primer of the OLR1 transcriptome is shown in SEQ ID NO.2: 5′-GCTTGCTCTTGTGTTAGGAGGT-3′.

[0008] The present invention also provides a vascular inflammation-related protein marker for auxiliary diagnosis or detection of cerebral aneurysm, wherein the protein marker is a combination of OLR1 protein, IL6 protein, PTX3 protein and LPL protein.

[0009] The present invention also provides a vascular inflammation-related protein marker for auxiliary diagnosis or detection of cerebral aneurysm, wherein the protein marker is a combination of OLR1 protein, IL6 protein and PTX3 protein.

[0010] Further, the above-mentioned vascular inflammation-related protein marker is used in the preparation of a kit for auxiliary diagnosis or detection of cerebral aneurysm, the kit includes RT-qPCR quantitative amplification primers for OLR1 transcriptome, IL6 transcriptome and PTX3 transcriptome, the nucleotide sequence of the forward amplification primer of the OLR1 transcriptome is shown in SEQ ID NO.1: 5′-TTGCCTGGGATTAGTAGTGACC-3′, the nucleotide sequence of the reverse amplification primer of the OLR1 transcriptome is shown in SEQ ID NO.2: 5′-GCTTGCTCTTGTGTTAGGAGGT-3′; the nucleotide sequence of the forward amplification primer of the IL6 transcriptome is shown in SEQ ID NO.3: 5′-TAGAGTACCTCCAGAACAGATT-3′, the nucleotide sequence of the reverse amplification primer of the IL6 transcriptome is shown in SEQ ID NO.4: 5′-AATAGTGTCCTAACGCTCATAC-3′; the nucleotide sequence of the forward amplification primer of the PTX3 transcriptome is shown in SEQ ID NO. NO.5 is shown as: 5′-GCATAATAGGAACACTTGAGAC-3′, and the nucleotide sequence of the reverse amplification primer of the PTX3 transcriptome is shown as SEQ ID NO.6: 5′-CTGACAGAGACACAGCATT-3′.

[0011] Compared with the prior art, the advantages of the present invention are: the present invention is a vascular inflammation-related protein marker for auxiliary diagnosis of cerebral aneurysms and its application, and discloses IL6, PTX3, OLR1, and LPL and their combined markers for auxiliary diagnosis of cerebral aneurysms for the first time. The transcriptome and protein levels of IL6, PTX3, OLR1 and LPL are expressed in the blood of patients with cerebral aneurysms. The mechanism may be that these vascular inflammation-related proteins continue to be highly expressed after inflammatory damage to the blood vessels, and participate in the pathological development process of cerebral aneurysms by increasing the secretion level of inflammatory factors. Therefore, the detection kit based on the detection of OLR1 markers, IL6, PTX3, OLR1 and LPL combined markers, and the transcriptome / protein expression level of IL6, PTX3 and OLR1 combined markers can conveniently and quickly realize the detection of cerebral aneurysms at the molecular level, with high detection efficiency, high sensitivity, and strong pertinence, which is conducive to the early detection and timely treatment of patients with cerebral aneurysms. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Figure 2 is the differential expression diagram of all vascular inflammation-related biomarkers between the cerebral aneurysm group and the control group, where A is the volcano diagram of 92 inflammation-related biomarkers, and B is the expression level difference diagram of 11 inflammation-related protein markers, *P<0.05, **P<0.01, ***P<0.001;

[0013] Figure 2 GO and KEGG enrichment analysis diagrams were performed for differentially expressed vascular inflammation-related proteins, where A is the top 20 GO enrichments based on the background of all annotated proteins, and B is the top 20 KEGG enrichment pathways based on the background of all annotated proteins;

[0014] Figure 3 Correlation heat map between differentially expressed vascular inflammation-related proteins in patients with cerebral aneurysms, *P<0.05, **P<0.01, ***P<0.001;

[0015] Figure 4 Figure 2 is a heat map of the correlation between differentially expressed vascular inflammatory proteins and clinical characteristics, where A is the correlation between the 11 inflammatory proteins and clinical characteristics in all groups, B is the correlation between the 11 inflammatory proteins and clinical characteristics in the cerebral aneurysm group, and C is the correlation between the 11 inflammatory proteins and clinical characteristics in the control group, *P<0.05, **P<0.01, ***P<0.001;

[0016] Figure 5The ELISA test results of PTX3 protein, IL6 protein, OLR1 protein and LPL protein in the plasma of the control group and the cerebral aneurysm group, where A is PTX3, B is IL6, C is OLR1, and D is LPL, *P<0.05, **P<0.01, ***P<0.001;

[0017] Figure 6 This is the ROC curve analysis diagram of the early diagnosis value of cerebral aneurysm verified by ELISA of PTX3 protein, IL6 protein, OLR1 protein and LPL protein and their combination;

[0018] Figure 7 The relative expression results of RT-qPCR of PTX3, IL6 and OLR1 transcriptome in the control group and blood leukocytes; A is PTX3, B is IL6, C is OLR1, ****P<0.0001;

[0019] Figure 8 ROC curve diagram for RT-qPCR validation of PTX3, IL6 and OLR1 transcriptome expression levels in blood leukocytes. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below with reference to the accompanying drawings.

[0021] Specific example 1: Screening for vascular inflammation-related proteins in cerebral aneurysms.

[0022] 1. Clinical data of volunteers in the screening phase: This study collected volunteers from the Department of Neurosurgery, the First Affiliated Hospital of Ningbo University, including 30 patients with cerebral aneurysms diagnosed by cerebral angiography (53.23±7.12, 15 males), and 29 control groups (53.27±6.96, 15 males) who were clinically confirmed to have no cerebral aneurysm lesions. The case group and the control group were strictly matched one to one in terms of gender and age, and fasting venous blood samples were collected from all participants for the detection of general biochemical indicators such as blood lipids and blood sugar and the detection of nucleic acid content in the blood. Their clinical data including gender, age, history of smoking and drinking, hypertension, and the results of routine blood biochemical tests were recorded. The clinical data of the first batch of cerebral aneurysm cases and control groups used for screening differential proteins are shown in Table 1 below.

[0023] 2. Plasma extraction and biochemical analysis: After obtaining informed consent from all participants, 4 mL of peripheral venous blood was collected within 6 hours after admission and placed in an anticoagulation tube. The upper plasma layer and the middle white blood cell layer were carefully extracted after centrifugation at 3000 rpm for 15 minutes at 4°C. Routine biochemical indicators, including blood glucose, total cholesterol, triglycerides, high-density lipoprotein, low-density lipoprotein, apolipoprotein A, apolipoprotein B, and apolipoprotein E, were examined using an automatic biochemical analyzer (Olympus AU2700, Japan). The results are shown in Table 1.

[0024] Table 1. Comparison of clinical data between the cerebral aneurysm case group and the control group in the first screening phase

[0025]

[0026] As shown in Table 1, the concentrations of apolipoprotein B and glucose in the cerebral aneurysm group were higher than those in the control group (P<0.05). There were no statistical differences in age, gender distribution, total cholesterol, high-density lipoprotein, low-density lipoprotein, apolipoprotein A and apolipoprotein E between the case group and the control group (P>0.05).

[0027] 3. Analysis of vascular inflammation-related proteins in cerebral aneurysms by ultrasensitive multiplex targeted protein detection analysis (abbreviated as Olink): According to the manufacturer's guidelines, plasma samples from 30 patients with cerebral aneurysms and 29 control patients were analyzed using the Olink® target 92 CVD II panel (Olink Proteomics AB, Sweden). This technology involves highly specific binding of target proteins to antibody probes labeled with dual oligonucleotides, followed by detection and quantification using a microfluidic real-time PCR instrument (Biomark HD, USA), and the final detection readout is displayed as a standardized protein expression value, which is used for various biological analyses after log2 transformation.

[0028] This study analyzed the expression levels of 92 vascular inflammation-related proteins in the Olink CVD-II group. Figure 1As shown in middle A, there were 11 differentially expressed inflammation-related proteins between the cerebral aneurysm group and the control group. Eight proteins were upregulated in the cerebral aneurysm group, including interleukin-6 (IL6), pentraxin-related protein 3 (PTX3), hepatitis A virus cellular receptor 1 (KIM1), carcinoembryonic antigen-related cell adhesion molecule 8 (CEACAM8), OLR1, interleukin-1 receptor antagonist (IL-1ra), angiopoietin-1 receptor (TIE2), and programmed cell death 1 ligand 2 (PD-L2). In contrast, three proteins were downregulated in the cerebral aneurysm group, including lipoprotein lipase (LPL), fatty acid binding protein 2 (FABP2), and interleukin-27 (IL-27). The remaining 81 vascular inflammation-related proteins were partially overlapped and not differentially expressed, so they are not shown in detail. Figure 1 The scatter plot in center B further illustrates the fold change difference of differential protein expression between the case group and the control group. Table 2 shows the detailed information of 11 differential proteins between the cerebral aneurysm case and control groups.

[0029] Table 2 shows the detailed information of vascular inflammatory proteins that were significantly changed between the cerebral aneurysm group and the control group

[0030]

[0031] Note: The fold change (FC) between the cerebral aneurysm group and the control group was calculated as log2 value. The p value (P<0.05) was calculated using t-test, indicating that there was a significant difference in the protein.

[0032] 4. Enrichment analysis and correlation analysis of differentially expressed proteins: When analyzing the differentially expressed protein (DEP) set between the cerebral aneurysm group and the control group, we used R software (Version 4.1.3) using the R package “Olink® Analyze” to determine the significant differentially expressed proteins. We used the ggplot2 package in R software for data visualization and analysis, generating heat maps and volcano maps to visualize the data. In addition, we used the versatile data visualization package ggplot2 in R software to perform gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. In addition, in order to study the relationship between the protein expression patterns of the two groups, Spearman correlation analysis was performed. We constructed a protein-protein interaction (PPI) network for the differentially expressed proteins. Subsequently, we used igraph [1.4.1] and ggraph [2.1.0] to visualize these networks. Finally, the ROC curve was generated using the ROCR package, and two or more indicators were combined using Logistic regression analysis. The area under the curve (AUC) value of the combined diagnosis was displayed in the legend in the lower right corner to evaluate the classification performance of the data.

[0033] Functional enrichment analysis was used to elucidate the potential functions of differentially expressed proteins in the plasma of cerebral aneurysms and control groups. Figure 2 As shown in Figure A, GO enrichment analysis showed that the differentially expressed proteins were enriched in pathways such as leukocyte migration, immune response, triglyceride metabolism, lipoprotein metabolism, acute phase response, and regulation of T cell proliferation. Figure 2 As shown in Figure B, KEGG enrichment analysis showed that the differentially expressed inflammation-related protein factors were mainly concentrated in pathways such as PPAR signaling, HIF-1 signaling, cytokine-cytokine interaction, and PI3K-Akt signaling. In addition, PPI network analysis was performed on the differentially expressed inflammation-related proteins, such as Figure 3 The results show that OLR1 has the highest score, indicating that it plays a vital role in the pathogenesis of IA. We also found that LPL is negatively correlated with IL-1ra, IL6, OLR1 and CEACAM8, FABP2 and TIE2, while other protein factors are positively correlated, and CEACAM8 and OLR1 show the most significant positive correlation. These different factors may be interrelated and play an important role in the occurrence of cerebral aneurysm disease or poor prognosis.

[0034] 5. Correlation between different inflammatory factors and clinical characteristics: We performed correlation analysis on the 11 differentially expressed proteins screened out with all groups, IA group and control group. As shown in Figure 4A, in all participants, apolipoprotein B was strongly correlated with IL-1ra (P<0.001), and TIE2 was positively correlated with both low-density lipoprotein and apolipoprotein B levels (P<0.01). It is worth noting that glucose concentration was positively correlated with IL-6 (P<0.01) and positively correlated with IL-1ra, OLR1, KIM-1 and PD-L2 (P<0.05). In contrast, apolipoprotein A was positively correlated with IL-27 (P<0.05), while triglycerides were positively correlated with IL-1ra (P<0.05) and negatively correlated with IL-27 (P<0.05). Gender was negatively correlated with CEACAM8 (P<0.05). As shown in Figure 4B, in the IA cohort, IL-1ra was positively correlated with triglyceride (P<0.01), apolipoprotein B was positively correlated with IL-1ra (P<0.05), glucose was positively correlated with IL-6 (P<0.05), and apolipoprotein E was positively correlated with TIE2 (P<0.05). In addition, gender was negatively correlated with IL-1ra (P<0.05), OLR1 (P<0.05), and CEACAM8 (P<0.01). Apolipoprotein E was negatively correlated with FABP2 (P<0.01) and LPL (P<0.05) (P<0.05). High-density lipoprotein was negatively correlated with IL-1ra (P<0.05). In contrast, as shown in Figure 4C, in the control group, apolipoprotein A was positively correlated with LPL and PD-L2 (P < 0.05), and high-density lipoprotein and IL-27 were positively correlated (P < 0.05), while age was negatively correlated with TIE2 (P < 0.05), and triglycerides were negatively correlated with IL-27 (P < 0.05). These findings suggest a complex interdependence between the examined biomarkers and clinical attributes.

[0035] In summary, we collected blood from 59 patients with cerebral aneurysm and a control group, and used Olink ultra-sensitive multiple targeted protein detection and analysis technology to detect 92 vascular inflammation-related proteins in the patients' plasma. A total of 11 inflammation-related proteins were found to be differentially expressed between the two groups of patients, namely IL6, PTX3, LPL, KIM1, CEACAM8, OLR1, IL-1ra, FABP2, TIE2, IL-27, and PD-L2.

[0036] Specific Example 2: Verify the diagnostic value of the vascular inflammation-related proteins screened in Specific Example 1 for cerebral aneurysms.

[0037] To further clarify the accuracy of Olink protein group detection, we further collected the second batch of validation set clinical cases and control group samples, a total of 30 pairs of IA patients and non-IA patients hospitalized in neurosurgery matched by gender and age as the control group. We selected the four proteins IL6, PTX3, OLR1 and LPL with the most significant protein expression differences between the case group and the control group, and used enzyme-linked immunosorbent assay (ELISA) to verify the differences in protein expression in patient plasma.

[0038] Table 3 is a comparison of clinical data between the cerebral aneurysm case group and the control group in the second validation phase

[0039]

[0040] As shown in Table 3, the plasma levels of total cholesterol (P = 0.013) and apolipoprotein E (P = 0.043) in the IA group were significantly lower than those in the control group. Figure 5 As shown in Figures A, B, C, and D, the ELISA results showed that the expression patterns of IL-6, PTX3, and OLR1 in the plasma of IA patients were significantly higher than those in the control group, while the expression pattern of LPL was significantly lower than that in the control group. Figure 6 As shown, ROC diagnostic analysis showed that the AUC of PTX3 was 0.90, the best sensitivity was 86.20%, and the specificity was 90.00%; the area under the curve (AUC) of IL-6 was 0.786, the best sensitivity was 73.33%, and the specificity was 90.00%; the AUC of OLR1 was 0.754, the best sensitivity was 70.00%, and the specificity was 76.66%; the AUC of LPL was 0.717, the best sensitivity was 76.66%, and the specificity was 73.33%. We combined the four predictive indicators and found that the combined AUC value was 0.922, the best sensitivity was 93.10%, and the specificity was 90.00%. These results emphasize the potential of IL6, PTX3, OLR1, and LPL as diagnostic biomarkers for IA, and also demonstrate that the diagnostic effect can be improved through multi-marker combination.

[0041] Specific example three: Real-time quantitative polymerase chain reaction RT-qPCR verification experiment was expanded for cases and control groups.

[0042] According to the ELISA test results in the specific example 2, RT-qPCR was further used to verify the expression of the mRNA levels of the three related inflammatory factors IL-6, PTX3 and OLR1 with AUC>0.75 in the patient's blood leukocytes. Total RNA was isolated from the blood buffy coat sample using TRIzol reagent. Subsequently, cDNA was synthesized from the RNA template using a cDNA reverse transcription kit (TransGenBiotech, China). RT-qPCR was performed on the Roche LightCycler 480 system using the SYBR Green SuperMix kit (TransGenBiotech, China). RT-qPCR specific primers were created using Primer6 software (Premier Biosoft, USA) for RT-qPCR quantitative analysis. The primer sequences are as follows:

[0043] The nucleotide sequence of the forward amplification primer of the OLR1 transcriptome is shown in SEQ ID NO.1: 5′-TTGCCTGGGATTAGTAGTGACC-3′, and the nucleotide sequence of the reverse amplification primer of the OLR1 transcriptome is shown in SEQ ID NO.2: 5′-GCTTGCTCTTGTGTTAGGAGGT-3′;

[0044] The nucleotide sequence of the forward amplification primer of the IL6 transcriptome is shown in SEQ ID NO.3: 5′-TAGAGTACCTCCAGAACAGATT-3′, and the nucleotide sequence of the reverse amplification primer of the IL6 transcriptome is shown in SEQ ID NO.4: 5′-GCTTGCTCTTGTGTTAGGAGGT-3′;

[0045] The nucleotide sequence of the forward amplification primer of the PTX3 transcriptome is shown in SEQ ID NO.5: 5′-GCATAATAGGAACACTTGAGAC-3′, and the nucleotide sequence of the reverse amplification primer of the PTX3 transcriptome is shown in SEQ ID NO.6: 5′-CTGACAGAGACACAGCATT-3′;

[0046] The nucleotide sequence of the internal reference gene ACTB forward amplification primer is shown in SEQ ID NO.7: 5′-ATTGCCGACAGGATGCAGA-3′, and the nucleotide sequence of the internal reference gene ACTB reverse amplification primer is shown in SEQ ID NO.8: 5′-CAGGAGGAGCAATGATCTTGAT-3′; 2 −△△Ct The relative expression of mRNA was analyzed by the method.

[0047] The results are as follows Figure 7As shown in Figure 2, the RT-qPCR results were consistent with the ELISA results. Figure 7 As shown in Figures A, B, and C, compared with the control group, the mRNA expression levels of PTX3 (P<0.001), IL6 (P<0.01), and OLR1 (P<0.001) in the blood leukocytes of patients with cerebral aneurysm were significantly increased. Figure 8 As shown, the results of ROC curve analysis showed that the IL6 transcriptome (AUC=1.00), PTX3 transcriptome (AUC=1.00) and KIM1 transcriptome (AUC=1.00) in the blood leukocytes of patients with cerebral aneurysm all had very high diagnostic values.

[0048] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A vascular inflammation-related protein marker for assisting diagnosis or detection of cerebral aneurysms, characterized in that: The protein marker is a combination of OLR1 protein, IL6 protein, PTX3 protein and LPL protein.

2. A vascular inflammation-related protein marker for assisting diagnosis or detection of cerebral aneurysms, characterized in that: The protein marker is a combination of OLR1 protein, IL6 protein and PTX3 protein.

3. Use of a reagent for detecting the vascular inflammation-related protein marker according to claim 2 in preparing a kit for auxiliary diagnosis or detection of cerebral aneurysms, characterized in that: The kit includes RT-qPCR quantitative amplification primers for OLR1 transcriptome, IL6 transcriptome and PTX3 transcriptome, The nucleotide sequence of the forward amplification primer of the OLR1 transcriptome is shown in SEQ ID NO.1: 5′-TTGCCTGGGATTAGTAGTGACC-3′, and the nucleotide sequence of the reverse amplification primer of the OLR1 transcriptome is shown in SEQ ID NO.2: 5′-GCTTGCTCTTGTGTTAGGAGGT-3′; The nucleotide sequence of the forward amplification primer of the IL6 transcriptome is shown in SEQ ID NO.3: 5′-TAGAGTACCTCCAGAACAGATT-3′, and the nucleotide sequence of the reverse amplification primer of the IL6 transcriptome is shown in SEQ ID NO.4: 5′-AATAGTGTCCTAACGCTCATAC-3′; The nucleotide sequence of the forward amplification primer of the PTX3 transcriptome is shown in SEQ ID NO.5: 5′-GCATAATAGGAACACTTGAGAC-3′, and the nucleotide sequence of the reverse amplification primer of the PTX3 transcriptome is shown in SEQ ID NO.6: 5′-CTGACAGAGACACAGCATT-3′.

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