Stress injury biomarker, screening method, detection reagent for detecting biomarker and application

Abstract

The invention provides a 3 / 4 stress injury biomarker and a screening method thereof, and belongs to the technical field of biomarkers. Through combined application of metabonomics, transcriptomics and proteomics, biomarkers of arginine, succinic acid and TRPC6 which are differentially expressed in wound tissue of a patient suffering from stress injury in the 3 / 4 period are found for the first time. The invention also provides a detection reagent for detecting the level of the biomarker in a sample to be detected, and the detection reagent is used for preparing a 3 / 4 stage stress injury diagnosis product. By coupling metabonomics-transcriptomics-proteomics means, high-sensitivity and full-coverage screening of 3 / 4 stage stress injury wound tissue biomarkers is realized, and various specific biomarkers closely associated with 3 / 4 stage stress injury are identified; and a better way and method are provided for analysis of the occurrence and development mechanism of the 3 / 4 stage stress injury and development of early prevention and treatment means.

Description

Technical Field

[0001] This invention belongs to the field of biomarker technology, specifically relating to pressure injury biomarkers, screening methods, and detection reagents and applications for detecting biomarkers. Background Technology

[0002] Pressure injury (PI), also known as pressure ulcer or bedsore, refers to the pathological process of tissue ulceration and necrosis caused by prolonged pressure on local tissues, leading to obstructed blood circulation. According to the latest edition of the "Clinical Practice Guidelines for the Prevention and Treatment of Pressure Ulcers / Pressure Injuries," pressure ulcers are classified into four stages based on the depth of tissue damage. Stages 3 / 4 PI are defined as severe pressure ulcers, characterized by full-thickness skin loss, and in severe cases, tendons or bone tissue may be exposed. The depth and extent of the wound are significantly increased, falling into the category of refractory chronic wounds. This condition is characterized by a prolonged course, complex treatment, high medical expenses, and stringent nursing requirements, seriously threatening patients' quality of life and placing a significant burden on the social medical security system. Notably, in recent years, it has become a research focus in geriatric medicine and wound repair, and related research has significant clinical value in improving prognosis and optimizing outcomes.

[0003] Currently, risk assessment for stage 3 / 4 primary infectious disease (PI) mainly relies on clinical evaluation systems based on subjective symptoms, such as the widely used Braden scale. These methods stratify risk by scoring parameters such as patient sensory perception, activity level, and nutritional status, but they have limitations including subjectivity, time lag, and lack of dynamic monitoring.

[0004] In recent years, some studies have attempted to explore biomarkers related to stage 3 / 4 primary inflammatory disease (PI) to improve predictive objectivity, such as detecting serum inflammatory factors IL-6, CRP, or tissue hypoxia-inducible factor HIF-1α. However, these single biochemical indicators suffer from limitations such as insufficient sensitivity, impaired specificity, and incomplete mechanistic coverage. Therefore, developing an early warning system based on a combination of multidimensional biomarkers has become a key research direction for overcoming the bottlenecks in the prevention and treatment of stage 3 / 4 PI, and has significant clinical value. Summary of the Invention

[0005] To overcome the problems existing in the prior art, this invention provides pressure injury biomarkers, screening methods, and detection reagents and applications for detecting biomarkers. By integrating metabolomics, transcriptomics, and proteomics technologies, a combination of biomarkers capable of predicting the risk of stage 3 / 4 PI is obtained. The invention systematically mines and verifies specific differential biomarkers closely associated with the progression of pressure injury, exhibiting high specificity, sensitivity, and accuracy, and providing a scientific basis for clinical diagnosis and targeted therapy.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] (i) The present invention provides a biomarker for pressure injury, wherein the biomarker includes at least one of arginine, succinic acid and TRPC6.

[0008] Furthermore, the pressure injury is a stage 3 / 4 pressure injury.

[0009] (ii) The present invention also provides a detection reagent for detecting the level of the biomarker as described in claim 1 in a sample to be tested.

[0010] (III) The present invention also provides a test kit, wherein the test kit includes the test reagents described above.

[0011] (iv) The present invention also provides the application of the above-described detection reagents in the preparation of diagnostic products for stage 3 / 4 pressure injuries.

[0012] (v) The present invention also provides a method for screening the above-mentioned biomarkers of pressure injury, wherein arginine and succinic acid are obtained by screening using full-spectrum metabolic LCMS omics technology; and TRPC6 is obtained by screening using reference transcriptomics technology and / or Pro DIA quantitative proteomics technology.

[0013] Furthermore, the process of screening arginine and succinic acid using full-spectrum metabolic LCMS omics technology is as follows: the wound tissue of stage 3 / 4 pressure injury and the normal tissue next to the wound are pretreated, the metabolites are separated, and the contents of arginine and succinic acid in the metabolites of the wound tissue and the normal tissue next to the wound are screened out. These are biomarkers of pressure injury.

[0014] Further, the pretreatment process is as follows: add pre-cooled homogenization buffer (methanol / water mixture) to the tissue, use a grinder to completely break the tissue, centrifuge, take the supernatant, remove impurities and cell debris, add pre-cooled extraction solvent (methanol:acetonitrile:water = 2:2:1), vortex mix, sonicate on ice for 20 min, centrifuge, take the supernatant, and freeze dry.

[0015] Furthermore, after pretreatment, the metabolites are separated by chromatography. Specifically, the chromatographic conditions are as follows: Column: C18 reversed-phase column for metabolite separation; Mobile phase: Positive ion mode, phase A (water + 0.1% formic acid), phase B (acetonitrile + 0.1% formic acid); Negative ion mode, phase A (water + 5mM ammonium acetate), phase B (acetonitrile); Gradient elution (5-95% phase B, 20-30 min), flow rate 0.3 mL / min, achieving efficient separation of complex metabolites.

[0016] Furthermore, after separating the metabolites, they were analyzed by mass spectrometry. Mass spectrometry conditions: instrument: high-resolution mass spectrometer; ionization mode: electrospray ionization (ESI), positive / negative ion mode switching; scan range: m / z 50-1500, resolution ≥30,000; quality control (QC): QC samples were inserted every 10 samples (all samples were mixed) to monitor instrument stability.

[0017] Further, following mass spectrometry analysis, data processing and analysis were performed: peak extraction and alignment, using software to extract mass spectrometry peaks and correct for retention time drift; denoising and normalization, removing background noise, performing data correction using QC samples, and normalizing the total peak area; metabolite annotation, based on precise mass numbers and secondary mass spectrometry fragments, matching databases (HMDB, METLIN, KEGG); statistical analysis, screening differentially expressed metabolites based on the OPLS-DA model, combined with t-tests / ANOVA (p<0.05), and variable importance projection VIP values ​​(VIP>1), to screen for significantly differentiating metabolites specific to stage 3 / 4 pressure injury.

[0018] Furthermore, the process of screening TRPC6 using reference transcriptomics technology is as follows: total RNA is extracted from wound tissue of stage 3 / 4 pressure injury and normal tissue adjacent to the wound, a strand-specific library is constructed, paired-end sequencing is performed, and TRPC6 is screened as a differentially expressed gene in the wound tissue and normal tissue adjacent to the wound.

[0019] Furthermore, total RNA was extracted using the TRIzol method, purity was detected by Nanodrop, and integrity was assessed using Agilent Bioanalyzer.

[0020] Furthermore, after extracting total RNA, eukaryotic mRNA was enriched, fragmented, and then cDNA was synthesized. A strand-specific library was constructed, and paired-end sequencing was performed using the Illumina NovaSeq 6000 platform to ensure comprehensive coverage of gene expression profiles.

[0021] Further, after paired-end sequencing, data processing and analysis were performed: after removing low-quality reads and adapters, an expression matrix was generated, and the DESeq2 algorithm was used to analyze the gene expression differences between wound and normal tissues. Combined with the characteristics of wound staging, specific differentially expressed genes in stage 3 / 4 pressure injury wound tissues were screened.

[0022] Furthermore, the process of screening TRPC6 using Pro DIA quantitative proteomics technology is as follows: extract total protein from stage 3 / 4 pressure injury wound tissue and adjacent normal tissue, convert the total protein into peptides, perform mass spectrometry analysis, and screen out the significantly differentially expressed protein in the wound tissue and adjacent normal tissue as TRPC6 protein.

[0023] Furthermore, after total protein extraction, the proteins were digested with trypsin to generate peptides, which were then desalted and lyophilized for concentration. Secondary fragment information was scanned using a Thermo Scientifi Orbitra Astra mass spectrometer. All mass spectrometry data were merged using the DIA-NN software to complete database retrieval of DIA mass spectrometry data and quantitative analysis of protein DIA. Based on log2 fold change (|FC|>1.5) and statistical significance (p<0.05, FDR correction), differentially expressed proteins (DEPs) specifically for stage 3 / 4 pressure injury were screened.

[0024] The beneficial effects of this invention are:

[0025] (1) This invention combines high-throughput metabolomics, transcriptomics, and proteomics analysis to reveal arginine, succinic acid, and TRPC6 as differential biomarkers in wound tissues and adjacent tissues of stage 3 / 4 PI patients; metabolomics pathway enrichment analysis screens for significantly differential metabolic pathways in wound tissues and adjacent tissues of stage 3 / 4 PI patients; transcriptomics KEGG enrichment analysis screens for significantly differential biological processes, molecular functions, and cellular components in wound tissues and adjacent tissues of stage 3 / 4 PI patients; transcriptomics GO enrichment analysis screens for significantly differential pathways in wound tissues and adjacent tissues of stage 3 / 4 PI patients; proteomics KEGG enrichment analysis screens for significantly differential biological processes, molecular functions, and cellular components in wound tissues and adjacent tissues of stage 3 / 4 PI patients; proteomics GO enrichment analysis screens for significantly differential pathways in wound tissues and adjacent tissues of stage 3 / 4 PI patients.

[0026] (2) This invention is the first to use tissue-derived arginine, succinic acid, and TRPC6 as specific combined biomarkers for stage 3 / 4 PI, providing a new direction for the early diagnosis and clinical treatment of stage 3 / 4 PI. The biomarkers (arginine, succinic acid, and TRPC6) screened in this invention can serve as diagnostic indicators for PI staging or potential therapeutic targets, providing a basis for the development of novel biological agents or metabolic intervention strategies. Attached Figure Description

[0027] Figure 1 This is a metabolic profile difference diagram between stage 3 / 4 PI wound tissue and adjacent normal tissue. A: KEGG enrichment analysis results; B: Difference in arginine content between the two wound tissues; C: Difference in succinic acid content between the two wound tissues.

[0028] Figure 2 This is a transcriptome showing the differences in transcriptomes between stage 3 / 4 PI wound tissue and adjacent normal tissue. A: KEGG (left) and GO (right) enrichment analysis results; B: Differences in TRPC6 mRNA expression between the two wound tissue groups.

[0029] Figure 3 This is a proteomic difference map between stage 3 / 4 PI wound tissue and adjacent normal tissue. A: KEGG (left) and GO (right) enrichment analysis results; B: Difference in TRPC6 protein expression between the two wound tissue groups. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the following embodiments are all conventional reagents, methods, and equipment in the art.

[0031] This invention provides biomarkers for stage 3 / 4 pressure injuries, including at least one of arginine, succinic acid, and TRPC6. Based on the identification of arginine, succinic acid, and TRPC6 as biomarkers for stage 3 / 4 pressure injuries, a diagnostic reagent or kit can be prepared to detect the levels of these three biomarkers. Diagnosis of stage 3 / 4 pressure injuries can be achieved by detecting the levels of arginine, succinic acid, and TRPC6 in the sample, providing a new direction for the early diagnosis and clinical treatment of stage 3 / 4 pressure injuries.

[0032] Example 1: Screening for specific metabolic biomarkers for phase 3 / 4 PIs using full-spectrum metabolic LCMS omics technology

[0033] The method for screening phase 3 / 4 PI-specific metabolic biomarkers using full-spectrum metabolic LCMS omics technology includes the following steps:

[0034] S1. Wound tissue and adjacent normal tissue (approximately 1 cm in diameter) were collected from 30 patients with stage 3 / 4 pressure injuries. After being flash-frozen in liquid nitrogen, the tissue was stored at -80°C. 50 mg of tissue sample was taken and 1 mL of pre-cooled methanol / water (4:1) mixture was added. The mixture was thoroughly broken up in a homogenizer (30 Hz, 5 min). The sample was centrifuged (12,000 rpm, 10 min, 4°C), and the supernatant was collected. 1 mL of pre-cooled extraction solvent (methanol:acetonitrile:water = 2:2:1) was added, and the mixture was vortexed and sonicated in an ice bath for 20 min. The sample was centrifuged again (12,000 rpm, 10 min, 4°C), and the supernatant was lyophilized.

[0035] S2. Chromatographic conditions: A C18 reversed-phase column (2.1 mm × 100 mm, 1.7 μm) was used, with mobile phases A (water + 0.1% formic acid) and B (acetonitrile + 0.1% formic acid), a gradient elution program of 0-20 min (5%-95% B phase), a flow rate of 0.3 mL / min, and a column temperature of 40 °C. Mass spectrometry conditions: High-resolution mass spectrometry (Thermo Q Exactive HF-X), positive / negative ion mode switching, scan range m / z 50-1500, resolution ≥30,000; QC samples (mixed samples) were inserted every 10 samples to monitor instrument stability.

[0036] S3. Peak extraction, alignment, and background subtraction were performed using Compound Discoverer 3.3 software, and the total peak area was normalized. Differences between groups were analyzed using the OPLS-DA model, and significantly different metabolites were screened using t-tests (p<0.05) and VIP values ​​(VIP>1). Metabolites were annotated based on the HMDB and KEGG databases, and arginine (m / z 175.1189, positive ion mode) and succinic acid (m / z 117.0193, negative ion mode) were identified as specific biomarkers. The identification of differentially differentiated metabolites between the two groups is as follows: Figure 1 As shown.

[0037] The contents of arginine and succinic acid in normal tissues were significantly downregulated compared with those in wound tissues (p<0.001), and the VIP values ​​were both >1.5, verifying their significant correlation with the progression of pressure injury.

[0038] Example 2: Screening of key differentially expressed genes in phase 3 / 4 PI using transcriptomics techniques

[0039] The method for screening key differentially expressed genes in phase 3 / 4 PI using transcriptomics technology includes the following steps:

[0040] S1. Wound tissue and adjacent normal tissue (approximately 1 cm in diameter) were collected from 12 patients with stage 3 / 4 pressure injuries. 100 mg of wound and normal tissue was taken, and total RNA was extracted using the TRIzol method. The purity was detected by Nanodrop (A260 / A280 = 1.8-2.0), and RNA integrity was assessed using Agilent Bioanalyzer 2100 (RIN value ≥ 7.0).

[0041] S2, strand-specific libraries were constructed using the NEBNext Ultra II RNALibrary Prep Kit and fragmented to 350bp; paired-end sequencing (PE150) was performed on the Illumina NovaSeq 6000 platform, with a sequencing depth of ≥6Gb per sample;

[0042] S3: Raw data underwent Trimmomatic re-extraction of low-quality reads and adapter sequences, and was aligned to the human reference genome (GRCh38) using Hisat2. Differentially expressed genes were screened using the DESeq2 algorithm (|log2FC|>1, p<0.05), and TRPC6 was identified as a key candidate gene by GO / KEGG enrichment analysis. Transcriptomic differences between the two groups are as follows: Figure 2 As shown.

[0043] TRPC6 is significantly highly expressed in wound tissue and is highly correlated with inflammatory response and calcium ion channel regulation pathways. Its expression level is significantly positively correlated with the progression of pressure injury, suggesting that it is involved in the pathological process of stage 3 / 4 PI.

[0044] Example 3: Screening of key differentially expressed proteins in phase 3 / 4 PIs using proteomics techniques

[0045] The method for screening key differentially expressed proteins in phase 3 / 4 PI using transcriptomics techniques includes the following steps:

[0046] S1. Wound tissue and adjacent normal tissue (approximately 1 cm in diameter) were collected from 12 patients with stage 3 / 4 pressure injuries. 50 mg of the wound and normal tissue were taken and RIPA lysis buffer was added to extract total protein, which was quantified by BCA method. 100 μg of protein was taken, reduced (5 mM DTT, 56℃ for 30 min), alkylated (15 mM IAA, protected from light for 30 min), digested with trypsin (1:50 w / w) at 37℃ for 16 h, and the peptides were desalted (C18 column) and then lyophilized and concentrated.

[0047] S2 uses a Thermo Orbitrap Astral mass spectrometer, DIA mode, with a first-stage scan range of m / z 350-1500 and a resolution of 120,000; and a second-stage scan range of m / z 200-2000 and a resolution of 30,000.

[0048] S3, DIA-NN software (v1.8.1) combined with the UniProt human protein database (2023 version) was used for database searching and quantification; screening criteria: |log2FC|>1.5, p<0.05 (FDR correction), finally identifying TRPC6 protein as significantly downregulated in wound tissue (p<0.0001). The proteomic differences between the two groups are as follows: Figure 3 As shown.

[0049] The results above show that the high expression of TRPC6 protein in wound tissue is consistent with the transcriptome data, confirming its reliability as a dual gene-protein marker and providing a molecular basis for targeted therapy.

[0050] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A biomarker for pressure injury, characterized in that, The markers include at least one of arginine, succinic acid, and TRPC6.

2. The pressure injury biomarker according to claim 1, characterized in that, The pressure injury is a stage 3 / 4 pressure injury.

3. A detection reagent, characterized in that, The detection reagent is used to detect the level of the biomarker described in claim 1 in the sample to be tested.

4. The use of the detection reagent according to claim 3 in the preparation of diagnostic products for stage 3 / 4 pressure injuries.

5. A test kit, characterized in that, The test kit includes the test reagent as described in claim 3.

6. The method for screening pressure injury biomarkers according to claim 1, characterized in that, The arginine and succinic acid were obtained by screening using full-spectrum metabolic LCMS omics technology. The TRPC6 was obtained through screening using parametric transcriptomics and / or Pro DIA quantitative proteomics.

7. The method for screening biomarkers of pressure injury according to claim 6, characterized in that, The process of screening arginine and succinic acid using full-spectrum metabolic LCMS omics technology is as follows: Pretreatment of stage 3 / 4 pressure injury wound tissue and adjacent normal tissue, separation of metabolites, and screening of the metabolites from the wound tissue and adjacent normal tissue revealed significant differences in the content of arginine and succinic acid, which are biomarkers of pressure injury.

8. The method for screening biomarkers of pressure injury according to claim 1, characterized in that, The process of screening TRPC6 using parametric transcriptomics technology is as follows: Total RNA was extracted from wound tissues of stage 3 / 4 pressure injuries and adjacent normal tissues. A strand-specific library was constructed, and paired-end sequencing was performed to screen for TRPC6 as a differentially expressed gene in the wound tissues and adjacent normal tissues.

9. The method for screening biomarkers of pressure injury according to claim 1, characterized in that, The process of screening TRPC6 using Pro DIA quantitative proteomics technology is as follows: Total protein was extracted from wound tissues of stage 3 / 4 pressure injuries and adjacent normal tissues. The total protein was converted into peptides and analyzed by mass spectrometry. TRPC6 protein was identified as a significantly differentially expressed protein in the wound tissues and adjacent normal tissues.

Images