RA traditional Chinese medicine syndrome typing based on protein antibody chip technology and application

Abstract

According to the RA traditional Chinese medicine syndrome typing based on the protein antibody chip technology and application, the protein antibody chip technology is applied to RA traditional Chinese medicine syndrome typing for the first time, and high-throughput and multi-target detection is achieved. Key markers such as IL-6R and TIMP-2 are found, and the combination of the key markers can remarkably improve the syndrome distinguishing accuracy; compared with single ELISA detection, the protein antibody chip technology can analyze 40 inflammatory factors at the same time, and the efficiency is greatly improved. In combination with bioinformatics, molecular characteristics of the damp-heat blockage syndrome and the liver and kidney deficiency syndrome are disclosed; an objective laboratory basis is provided for traditional Chinese medicine differentiation of RA, and clinical precise typing and individualized treatment are assisted.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a method for TCM syndrome differentiation and application of RA based on protein antibody chip technology. Background Technology

[0002] The TCM syndrome differentiation of rheumatoid arthritis (RA) (such as damp-heat obstruction syndrome characterized by joint redness, swelling, heat and pain, red tongue with yellow and greasy coating, and slippery and rapid pulse; and liver and kidney deficiency syndrome characterized by dull joint pain, soreness and weakness of the waist and knees, pale tongue with white coating, and deep and thready pulse) currently lacks objective molecular marker support. Traditional syndrome differentiation mainly relies on the subjective experience of physicians in judging symptoms, tongue and pulse conditions, which has problems such as inconsistent diagnostic criteria and low consistency of diagnosis among different physicians (Kappa value is often <0.6). This can easily lead to misjudgment of syndrome or missed diagnosis of early cases, thus affecting the accurate selection of individualized treatment plans.

[0003] Existing inflammatory factor detection technologies (such as enzyme-linked immunosorbent assay, ELISA) have limitations due to their low throughput. A single test can typically analyze only one or a few inflammatory factors, making it difficult to simultaneously cover multiple key factors (such as pro-inflammatory factors, anti-inflammatory factors, and chemokines) in the complex inflammatory network of RA. This results in an inability to fully reveal the systemic differences in molecular mechanisms among different syndromes. 3. Protein antibody microarray technology, as a high-throughput detection method, can simultaneously quantify hundreds of protein molecules and has been widely used in biomarker screening in fields such as oncology and autoimmune diseases. However, in the field of TCM syndrome differentiation of RA, no research has yet used it for the discovery of specific molecular biomarkers for damp-heat obstruction syndrome and liver and kidney deficiency syndrome, or for differentiating the essence of the syndromes.

[0004] Furthermore, existing research on TCM syndromes related to RA mainly focuses on clinical symptom description or single factor detection, which has shortcomings such as small sample size (mostly <100 cases) and limited types of inflammatory factors detected (mostly <20 types). It has not yet systematically elucidated the characteristic differences in the expression profiles of inflammatory factors in the serum of patients with damp-heat obstruction syndrome and liver and kidney deficiency syndrome, nor has it clarified the clinical value of these differentially expressed factors in syndrome diagnosis, such as sensitivity and specificity, thus restricting the objectification and standardization of TCM syndrome differentiation. Summary of the Invention

[0005] To address the technical problems existing in the prior art, the present invention provides the following technical solution:

[0006] The purpose of this invention is to provide a TCM syndrome differentiation method for RA based on protein antibody chip technology, which solves the problem of strong subjectivity in traditional syndrome differentiation; by screening specific inflammatory factors (IL-6R, CXCL13, TIMP-2, etc.), an objective combination of syndrome differentiation markers is established; combined with bioinformatics analysis (GO / KEGG), the molecular mechanisms of syndrome differences are revealed, providing a basis for precision treatment.

[0007] On the one hand, a diagnostic kit based on protein antibody chip technology is provided, the diagnostic kit including a protein antibody chip, wherein the protein antibody chip is configured with the following differentially expressed inflammatory factors:

[0008] TIMP-2, wherein TIMP-2 serves as a diagnostic marker for liver and kidney deficiency syndrome;

[0009] or

[0010] A differentially expressed combination of IL-6R, CXCL13, and TIMP-2, wherein the differentially expressed combination of inflammatory factors serves as a diagnostic biomarker for damp-heat obstruction syndrome.

[0011] Preferably, the protein antibody chip is further configured with at least one of the following differentially expressed inflammatory factors:

[0012] TNF-RI, TNF-RII, or PDGF-BB.

[0013] Preferably, the protein antibody chip is GSH-INF-3 RayBiotech.

[0014] On the other hand, a method for TCM syndrome differentiation of rheumatoid arthritis based on the diagnostic kit using the protein antibody chip technology described above is provided, the method comprising the following steps:

[0015] (1) Protein antibody chip detection:

[0016] ① After the glass slide chip is equilibrated at room temperature for 20-30 minutes, it is dried for 1-2 hours;

[0017] ② Add 100 μL of diluent to each well for sealing treatment, and incubate on a shaker at room temperature for 0.5-3 hours;

[0018] ③ After removing the buffer, add 100 μL of RA peripheral serum sample diluted 1:2 and incubate overnight at 3-10℃;

[0019] ④ Clean the chip sequentially with cleaning solution I and cleaning solution II;

[0020] ⑤ Add 80 μL of the detection antibody mixture to each well, incubate on a shaker at room temperature for 1.5-4 hours, and then repeat step ④ for washing;

[0021] ⑥ Add 80 μL of Cy3-streptavidin to each well, incubate in the dark at room temperature for 0.5-3 hours, and then repeat step ④ for washing;

[0022] ⑦ Scan and detect fluorescence signals;

[0023] (2) Data analysis: Normalize the raw data and screen for differentially inflammatory factors with logFC > 1.2 or < 0.83 and P < 0.05;

[0024] (3) Syndrome classification: Based on the expression level of differential factors and bioinformatics analysis, the RA damp-heat obstruction syndrome and liver and kidney deficiency syndrome are distinguished.

[0025] Preferably, in step (1) ④, both washing solution I and washing solution II are diluted with deionized water to 20× concentrate, and the shaking intensity is high during washing, with each shaking lasting 10 seconds.

[0026] Preferably, the ELISA method is used to verify the differential factors in step (2), and the verification indicators include IL-6R, TIMP-2, and CXCL13.

[0027] Preferably, the differential inflammatory factors in step (2) include IL-6R, CXCL13, TIMP-2, TNF-RI, TNF-RII and PDGF-BB.

[0028] Preferably, the bioinformatics analysis in step (3) includes:

[0029] GO enrichment analysis involves biological processes such as chemotactic regulation, TNF receptor activity, and extracellular matrix tissue.

[0030] KEGG pathway analysis: involved JAK-STAT pathway, TNF pathway, and cytokine-receptor interaction pathway.

[0031] The beneficial effects of the technical solutions provided in the embodiments of the present invention include at least the following:

[0032] This invention is the first to apply protein antibody chip technology to TCM syndrome differentiation in rheumatoid arthritis (RA), achieving high-throughput, multi-target detection. Key biomarkers such as IL-6R and TIMP-2 were discovered, and their combination significantly improves the accuracy of syndrome differentiation. Compared to single ELISA detection, protein antibody chip technology can simultaneously analyze 40 inflammatory factors, greatly improving efficiency. Combined with bioinformatics, the molecular characteristics of damp-heat obstruction syndrome and liver-kidney deficiency syndrome are revealed; providing objective laboratory evidence for TCM syndrome differentiation of RA, and assisting in precise clinical classification and individualized treatment. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1This is a flowchart of the detection steps of a protein antibody chip provided in an embodiment of the present invention;

[0035] Figure 2 This is a schematic diagram of a GSH-INF-3 protein antibody chip detection index (40 inflammatory factors) provided in an embodiment of the present invention;

[0036] Figure 3 This is a diagram illustrating the interaction analysis of differentially expressed proteins provided in an embodiment of the present invention.

[0037] Figure 4 This is a schematic diagram of principal component analysis provided in an embodiment of the present invention;

[0038] Figure 5 This is a clustering analysis diagram provided in an embodiment of the present invention;

[0039] Figure 6 This is an enrichment histogram provided in an embodiment of the present invention;

[0040] Figure 7 This is a KEGG enrichment analysis bar chart provided in an embodiment of the present invention;

[0041] Figure 8 This is a correlation analysis diagram of inflammatory factors and disease indicators provided in an embodiment of the present invention;

[0042] Figure 9 This is an IL-6R and TIMP-2 ROC curve provided in an embodiment of the present invention. Detailed Implementation

[0043] The technical solution of the present invention will now be described with reference to the accompanying drawings.

[0044] In embodiments of the present invention, words such as "exemplarily," "for example," etc., are used to indicate that something is an example, illustration, or description. Any embodiment or design described as "exemplary" in the present invention should not be construed as being more preferred or advantageous than other embodiments or designs. Specifically, the use of the word "exemplary" is intended to present the concept in a concrete manner. Furthermore, in embodiments of the present invention, the meaning expressed by "and / or" can be both, or either one.

[0045] In the embodiments of this invention, the terms "image" and "picture" may sometimes be used interchangeably. It should be noted that, without emphasizing the distinction between them, they convey the same meaning. Similarly, the terms "of," "corresponding (relevant)," and "corresponding" may sometimes be used interchangeably. It should be noted that, without emphasizing the distinction between them, they convey the same meaning.

[0046] In this embodiment of the invention, sometimes a subscript such as W1 may be mistakenly written as a non-subscript form such as W1. When the difference is not emphasized, the meaning they express is the same.

[0047] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0048] This invention provides a TCM syndrome differentiation and application for RA based on protein antibody chip technology. The technical implementation steps, principles, and experimental analysis results of this invention will be described below.

[0049] (I) Technical Solution

[0050] 1. Collection, aliquoting, and preservation of serum samples from RA patients.

[0051] (1) After standard aseptic disinfection, 2 mL of peripheral whole blood sample was collected from each subject by venipuncture; (2) After the blood sample was thoroughly mixed, it was immediately placed in a centrifuge for centrifugation at 3000 rpm for 10 min; (3) After centrifugation, the supernatant was accurately aspirated using a sterile pipette and dispensed into 1.5 mL EP tubes that had been pre-sterilized; (4) The EP tubes were numbered and labeled and then stored at -80℃ for long-term cryopreservation. During storage, the temperature fluctuation range was strictly controlled within ±2℃.

[0052] 2. Detection Method

[0053] The testing process mainly consists of the following steps:

[0054] (1) Drying treatment of glass slide chips

[0055] Remove the glass chip from the box and let it sit at room temperature for 20-30 minutes before opening the packaging. Place the chip in a vacuum drying apparatus or at room temperature for 1-2 hours to ensure it is completely dry.

[0056] (2) Chip operation process

[0057] ① Add 100 µL of diluent to each well and incubate on a shaker at room temperature for 1 hour to block the quantitative antibody chip.

[0058] ② After removing the buffer from each well, add 100 µL of serum sample diluted 1:2 and incubate overnight at 4°C.

[0059] ③ Clean the glass slides using the Thermo Scientific Wellwash Versa chip washer. The steps are as follows:

[0060] Step 1: Clean with 1× Washing Solution I, add 250 μL to each well, repeat the cleaning 10 times, shake for 10 seconds each time, set the shaking intensity to high, and dilute 20× Washing Solution I with deionized water.

[0061] Step 2: Switch to the 1× Wash Solution II channel for cleaning, add 250 μL to each well, clean 6 times, with the same shaking intensity as above, and dilute 20× Wash Solution II with deionized water.

[0062] ④ Incubation of the antibody mixture: Centrifuge the antibody complex, mix it with 1.4 mL of sample diluent, and centrifuge again quickly. Add 80 µL of the antibody to each well and incubate on a shaker at room temperature for 2 hours.

[0063] ⑤ Clean, and repeat step ③ to complete the cleaning process.

[0064] ⑥ Incubation of Cy3-Streptavidin: Centrifuge the Cy3-streptavidin tubes, mix thoroughly with 1.4 mL of sample dilution, and centrifuge again. Add 80 µL of Cy3-streptavidin to each well, wrap the slide with aluminum foil, and incubate on a shaker at room temperature for 1 hour in the dark.

[0065] ⑦ Clean, and repeat step ③ to complete the cleaning.

[0066] ⑧ Fluorescence detection: Fluorescence detection was performed using an InnoScan 300 scanner. The scanning parameters were a wavelength of 532 nm and a resolution of 10 µm.

[0067] (3) Data analysis: GSH-INF-3 data analysis software is used for data analysis (Figure 1: Protein chip detection steps).

[0068] 3. Detection indicators

[0069] The GSH-INF-3 RayBiotech protein antibody chip detects a total of 40 inflammatory markers: CXCL13, CCL11, MPIF-2, G-CSF, GM-CSF, TCA-3 / CCL1, CD54, IFN-γ, IL-1α, IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-6R, IL-7, IL-8, IL-10, IL-11, IL-12 p40, IL-12 p70, IL-13, IL-15, IL-16, IL-17A, CCL2, M-CSF, CXCL9, CCL3, CCL4, CCL15, PDGF-BB, CCL5, TIMP-1, TIMP-2, TNF-α, TNF-β, TNFRI, and TNFRII. Figure 2(GSH-INF-3 RayBiotech protein antibody chip detects 40 inflammatory factors).

[0070] 4. Screening and data analysis of differentially expressed inflammatory factors

[0071] (1) RayBiotech was used to perform normalization analysis on the raw data. The process included background correction, inter-chip normalization and normalization. First, the raw data was normalized in the application software. Then, the normalization was applied to the data through the limma data package (R / Bioconductor) for further analysis. The analysis method was modified t-statistics. Different proteins were screened using the adjusted p value (P value after BH method correction) and logFC (fold change in expression, base 2). The selection criteria were: logFC greater than 1.2 or less than 0.83, and P value or calibrated P value < 0.05.

[0072] (2) Validation method: ELISA to validate differential factors

[0073] (3) Bioinformatics: GO enrichment analysis (e.g., chemotactic regulation, TNF receptor activity), KEGG pathway analysis (e.g., JAK-STAT, TNF pathway); clinical correlation: Spearman correlation analysis; ROC curve assessment of diagnostic efficacy (AUC>0.7).

[0074] (II) Implementation Examples

[0075] Test samples: serum from 14 patients with damp-heat obstruction syndrome and 8 patients with liver and kidney deficiency syndrome of RA.

[0076] Research process: 1. Using protein antibody chip technology, the serum differential inflammatory factors of RA damp-heat obstruction syndrome and liver and kidney deficiency syndrome were obtained. The main characteristics were increased expression of IL-6R, CXCL13 and PDGF-BB in damp-heat obstruction syndrome and increased expression of TIMP-2, TNF-RI and TNF-RII in liver and kidney deficiency syndrome.

[0077] 2. Bioinformatics analysis: (1) GO enrichment analysis revealed 267 significantly enriched GO BPs. Based on the Count and P values, 20 were selected, specifically including regulation of peptide tyrosine phosphorylation, chemotactic regulation, negative regulation of response to external stimuli, monocyte migration, and apoptosis.

[0078] (2) GO enrichment analysis revealed 12 significantly enriched GO CCs, based on Count and P values, specifically tumor necrosis factor receptor activity, growth factor and receptor binding, and cytokine binding.

[0079] (3) GO enrichment analysis revealed 18 significantly enriched GO MFs, based on Count and P values, specifically vesicle cavities, secretory granule cavities, and extracellular matrix containing collagen.

[0080] (4) KEGG enrichment analysis showed that a total of 14 differentially enriched signaling pathways were significantly enriched, based on the Count and P values. Specifically, these pathways included the interaction between viral proteins and cytokines and cytokine receptors, the TNF pathway, the MAPK pathway, the JAK-STAT pathway, and the interaction between cytokines and cytokine receptors.

[0081] 3. ELISA was used for verification. The results showed that there were statistically significant differences in the expression of IL-6R, TIMP-2, and CXCL13 between RA with damp-heat obstruction syndrome and RA with liver and kidney deficiency syndrome. The expression levels of IL-6R and CXCL13 were higher in RA with damp-heat obstruction syndrome, while the expression level of TIMP-2 was higher in RA with liver and kidney deficiency syndrome (P<0.05).

[0082] 4. Spearman correlation analysis revealed an interaction between serum IL-6R, TIMP-2, and TNF-RII. TIMP-2 levels were negatively correlated with DAS28 (P<0.05), suggesting that TIMP-2 may be a serum biomarker related to RA disease activity.

[0083] 5. ROC analysis revealed that IL-6R and TIMP-2 levels have potential diagnostic value in differentiating between damp-heat obstruction syndrome and liver and kidney deficiency syndrome in rheumatoid arthritis. In particular, IL-6R had the largest area under the ROC curve (AUC>0.7), indicating that it is more likely to be a serum marker for the diagnosis of damp-heat obstruction syndrome in RA.

[0084] 1) Comparison of serum levels of related inflammatory factors in rheumatoid arthritis patients with damp-heat obstruction syndrome and those with liver and kidney deficiency syndrome.

[0085] A comparison of serum levels of relevant inflammatory factors between patients with RA (rheumatoid arthritis) and those with liver and kidney deficiency syndrome showed that, compared with those with liver and kidney deficiency syndrome, patients with damp-heat obstruction syndrome had higher levels of CXCL13, IL-6R, and PDGF-BB, and lower levels of TIMP-2, TNF-RI, and TNF-RII. The differences were statistically significant (P < 0.05) (see Table 1).

[0086] Table 1. Comparison of serum levels of related inflammatory factors in the two groups of rheumatoid arthritis with damp-heat obstruction syndrome and liver and kidney deficiency syndrome.

[0087]

[0088] 2) Protein-protein interaction analysis

[0089] PPIs refer to the binding of two or more proteins, typically intended to perform their biochemical functions. This study used the online tool STRING (http: / / string-db.org / ) to analyze the protein-protein interactions of six screened inflammatory factors, constructing a protein-protein interaction network. The results showed that, except for TIMP-2, the other five cytokines interacted with each other, forming a complex network. Figure 3 For example, there are three interactions between TNF-RII and IL-6R.

[0090] 3) Study on the functional enrichment characteristics of differential expression of inflammatory factors

[0091] 3.1) Principal Component Analysis

[0092] Principal Components Analysis (PCA) is an unsupervised method used to analyze serum samples from patients with rheumatoid arthritis (RA) of damp-heat syndrome and liver-kidney deficiency syndrome to observe whether there are differences in the expression of inflammatory factors detected by protein antibody microarrays between the two groups. A score plot is constructed using the two most important principal components representing the information variables in the matrix, with each point in the plot representing the expression of inflammatory factors in each sample. The PCA score plots of RA patients with damp-heat syndrome and liver-kidney deficiency syndrome show a certain degree of differentiation in the expression of inflammatory factors in the synovial fluid of RA patients with different syndromes. Figure 4 ).

[0093] 3.2) Cluster heatmap analysis

[0094] Differentially expressed inflammatory factors in the serum of RA patients with damp-heat obstruction syndrome and liver-kidney deficiency syndrome were normalized and then subjected to dendritic cluster analysis. Cluster heatmap analysis revealed significant differences in serum inflammatory factor expression between these two groups. Figure 5 ).

[0095] 3.3) GO and KEGG enrichment analysis

[0096] The results of GO biological process enrichment analysis showed that 267 significantly enriched GO BPs were found in patients with RA damp-heat obstruction syndrome and liver and kidney deficiency syndrome. Twenty BPs were selected based on count and p-value, as shown in Table 2. Figure 6As shown, specifically: regulation of glomerular mesangial cell proliferation, negative regulation of response to external stimuli, glomerular mesangial cell proliferation, regulation of apoptosis during morphogenesis, secretion of extracellular matrix components, regulation of apoptosis during development, positive regulation of chemotaxis, glomerular mesangial development, positive regulation of peptidyltyrosine phosphorylation, cell proliferation involved in kidney development, negative regulation of extracellular matrix tissues, pulmonary valve development, monocyte migration, apoptosis during morphogenesis, regulation of extracellular domain proteolysis of membrane proteins, regulation of peptidyltyrosine phosphorylation, regulation of chemotaxis, exogenous apoptosis signaling pathways, and glomerular vascular system development.

[0097] Table 2. GO-BP significantly enriched in RA damp-heat obstruction syndrome and liver-kidney deficiency syndrome

[0098] ID BP English name BP Chinese name BgRatio P GO:0072124 regulation of glomerular mesangial cell proliferation Regulation of glomerular mesangial cell proliferation 11 / 18888 <0.01 GO:0032102 negative regulation of response to external stimulus Negative regulation of response to external stimuli 456 / 18888 <0.01 GO:0072110 glomerular mesangial cell proliferation Glomerular mesangial cell proliferation 12 / 18888 <0.01 GO:1902337 regulation of apoptotic process involved in morphogenesis Regulation of apoptosis during morphogenesis 12 / 18888 <0.01 GO:0070278 extracellular matrix constituent secretion extracellular matrix component secretion 13 / 18888 <0.01 GO:1904748 regulation of apoptotic process involved in development Regulation of apoptosis during development 13 / 18888 <0.01 GO:0050921 positive regulation of chemotaxis Positive regulation of chemotaxis 142 / 18888 <0.01 GO:1901722 regulation of cell proliferation involved in kidney development Regulation of cell proliferation during kidney development 15 / 18888 <0.01 GO:0072109 glomerular mesangium development Glomerular mesangial development 17 / 18888 <0.01 GO:0050731 positive regulation of peptidyl-tyrosine phosphorylation Positive regulation of peptidyltyrosine phosphorylation 164 / 18888 <0.01 GO:0072111 cell proliferation involved in kidney development Cell proliferation involved in kidney development 22 / 18888 <0.01 GO:1903054 negative regulation of extracellular matrix organization Negative regulation of extracellular matrix tissue 22 / 18888 <0.01 GO:0003177 pulmonary valve development pulmonary valve development 23 / 18888 <0.01 GO:0071674 mononuclear cell migration Monocyte migration 206 / 18888 <0.01 GO:0060561 apoptotic process involved in morphogenesis Apoptosis during morphogenesis 25 / 18888 <0.01 GO:0051043 regulation of membrane protein ectodomain proteolysis Regulation of extracellular domain proteolysis of membrane proteins 27 / 18888 <0.01 GO:0050730 regulation of peptidyl-tyrosine phosphorylation Regulation of peptidyltyrosine phosphorylation 225 / 18888 <0.01 GO:0050920 regulation of chemotaxis Regulation of chemotaxis 229 / 18888 <0.01 GO:0097191 extrinsic apoptotic signaling pathway Exogenous apoptosis signaling pathway 230 / 18888 <0.01 GO:0072012 glomerulus vasculature development Glomerular vascular system development 30 / 18888 0.06

[0099] The results of GO cell component enrichment analysis showed that 12 significantly enriched GO CCs were found in patients with RA damp-heat obstruction syndrome and liver and kidney deficiency syndrome. These were screened based on count and p-value, as shown in Table 3. Figure 6 As shown, specifically: specific granules, membrane rafts, membrane microregions, secretory granule cavities, cytoplasmic vesicle cavities, vesicle cavities, collagen-containing extracellular matrix, tertiary granule cavities, specific granule cavities, platelet α-granule cavities, axons, etc.

[0100] Table 3. GO-CC significantly enriched in RA damp-heat obstruction syndrome and liver-kidney deficiency syndrome.

[0101]

[0102] The results of GO molecular functional enrichment analysis showed that 17 significantly enriched GO molecular functions were present in patients with RA damp-heat obstruction syndrome and liver and kidney deficiency syndrome. These were screened based on count and p-values, as shown in Table 4. Figure 6 The data includes, specifically: tumor necrosis factor receptor activity, death receptor activity, growth factor binding, cytokine binding, growth factor receptor binding, cytokine receptor binding, superoxide dismutase (SOD) oxidase activator activity, platelet-derived growth factor (PDGF) binding, PGF receptor binding, metalloendopeptidase inhibitor activity, CXCR chemokine receptor binding, fibroblast growth factor (FOD) binding, chemotactic activity, chemokine activity, CCR chemokine receptor binding, collagen binding, chemokine receptor binding, and tumor necrosis factor receptor activity. Each entry is sorted by its -log10 p-value, and the top 10 entries for each entry are displayed.

[0103] Table 4---GO-MF significantly enriched in RA damp-heat obstruction syndrome and liver and kidney deficiency syndrome

[0104]

[0105] KEGG enrichment analysis revealed 14 significantly enriched differentially expressed signaling pathways in patients with RA (rheumatoid arthritis) of damp-heat obstruction syndrome and liver-kidney deficiency syndrome. These pathways were further selected based on count and p-value, as shown in Table 5. Figure 7 The specific interactions shown include: the interaction between viral proteins and cytokines and cytokine receptors; cytokine-cytokine receptor interactions; adipokines signaling pathways; resistance to EGFR tyrosine kinase inhibitors; fluid shear stress and atherosclerosis; TNF signaling pathway in non-alcoholic fatty liver disease; Kaposi's sarcoma-associated herpesvirus infection; JAK-STAT signaling pathway; human immunodeficiency virus type 1 infection; human cytomegalovirus infection; coronavirus-COVID-19; MAPK signaling pathway; and the interaction between viral proteins and cytokines and cytokine receptors.

[0106] Table 5--- Significantly Different KEGG Enrichment Signaling Pathways Between RA Damp-Heat Obstruction Syndrome and Liver-Kidney Deficiency Syndrome

[0107]

[0108] 4. ELISA Results

[0109] Serum concentrations of IL-6R, TIMP-2, CXCL13, TNF-RI, TNF-RII, and PDGF-BB were detected using ELISA in the same batch of samples. A comparison of serum levels of related inflammatory factors in patients with RA (damp-heat obstruction syndrome) and those with liver-kidney deficiency syndrome showed that, compared with those with liver-kidney deficiency syndrome, patients with damp-heat obstruction syndrome had higher concentrations of CXCL13 and IL-6R and lower concentrations of TIMP-2, with statistically significant differences (P < 0.05) (see Table 6). Figure 8 Although there were no statistically significant differences among TNF-RI, TNF-RII, and PDGF-BB, the trend observed was consistent with the previous findings.

[0110] Table 6--- Serum levels of relevant inflammatory factors in patients with RA (damp-heat obstruction syndrome) and those with liver and kidney deficiency syndrome.

[0111]

[0112] (III) Correlation and Diagnostic Analysis

[0113] 1. Association analysis between inflammatory factors and clinical characteristics of RA patients

[0114] To explore whether serum inflammation levels are correlated with the severity of RA disease activity, and because RA-related clinical indicators (including DAS28, RF, anti-CCP antibody, CRP, and ESR) in the samples did not conform to a normal distribution according to the Shapiro-Wilk test, we performed a Spearman rank correlation test to assess their correlation with serum IL-6R, TIMP-2, CXCL13, PDGF-BB, TNF-RI, and TNF-RII levels. Serum TIMP-2 was negatively correlated with DAS28, while serum expression levels of IL-6R, TIMP-2, TNF-RI, and TNF-RII showed a correlation trend, as shown in Table 7. Figure 8 As shown.

[0115] Table 7---Correlation analysis of clinical factors and IL-6R, TIMP-2, BLC, PDGF-BB, TNF-RI, and TNF-RII

[0116]

[0117] Note: *P<0.05, **P<0.01.

[0118] 2. ROC curve analysis

[0119] ROC curve analysis was used to evaluate the diagnostic value of serum differentially expressed inflammatory factors for rheumatoid arthritis (RA) with damp-heat obstruction syndrome. The results showed that IL-6R and TIMP-2 levels could differentiate RA with damp-heat obstruction syndrome from liver and kidney deficiency syndrome. Figure 9 As shown in Table 8, the maximum area under the curve was found to be IL-6R (AUC: 0.813, 95%Cl: 0.569-0.932).

[0120] Table 8--- ROC Analysis of IL-6R and TIMP-2

[0121] Inflammatory factors AUC P 95% CI TIMP-2 0.795 0.024 0.594-0.995 IL-6R 0.813 0.017 0.569-0.932

[0122] In this embodiment, the above targets can also be replaced by the following: (1) In addition to IL-6R and TIMP-2, they can be extended to other differential factors (such as CXCL13 and TNF-RII). (2) They can be combined to detect RA-specific indicators such as rheumatoid factor and anti-CCP antibody to enhance the reliability of diagnosis.

[0123] Therefore, compared to single ELISA detection, the protein antibody chip technology of this invention can simultaneously analyze 40 inflammatory factors, significantly improving efficiency. Combined with bioinformatics, it reveals the molecular characteristics of damp-heat obstruction syndrome and liver-kidney deficiency syndrome, providing objective laboratory evidence for TCM syndrome differentiation of RA, and assisting in precise clinical subtyping and individualized treatment.

[0124] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A diagnostic kit based on protein antibody chip technology, characterized in that, The diagnostic kit includes a protein antibody chip, wherein the protein antibody chip is configured with the following differentially expressed inflammatory factors: TIMP-2, wherein TIMP-2 serves as a diagnostic marker for liver and kidney deficiency syndrome; or A differentially expressed combination of IL-6R, CXCL13, and TIMP-2, wherein the differentially expressed combination of inflammatory factors serves as a diagnostic biomarker for damp-heat obstruction syndrome.

2. The diagnostic kit based on protein antibody chip technology according to claim 1, characterized in that, The protein antibody chip is also equipped with at least one of the following differentially expressed inflammatory factors: TNF-RI, TNF-RII, or PDGF-BB.

3. The diagnostic kit based on protein antibody chip technology according to claim 1, characterized in that, The protein antibody chip is GSH-INF-3 RayBiotech.

4. A method for TCM syndrome differentiation of rheumatoid arthritis based on a diagnostic kit using protein antibody chip technology as described in any one of claims 1-3, characterized in that, The method includes the following steps: (1) Protein antibody chip detection: ① After the glass slide chip is equilibrated at room temperature for 20-30 minutes, it is dried for 1-2 hours; ② Add 100 μL of diluent to each well for sealing treatment, and incubate on a shaker at room temperature for 0.5-3 hours; ③ After removing the buffer, add 100 μL of RA peripheral serum sample diluted 1:2 and incubate overnight at 3-10℃; ④ Clean the chip sequentially with cleaning solution I and cleaning solution II; ⑤ Add 80 μL of the detection antibody mixture to each well, incubate on a shaker at room temperature for 1.5-4 hours, and then repeat step ④ for washing; ⑥ Add 80 μL of Cy3-streptavidin to each well, incubate in the dark at room temperature for 0.5-3 hours, and then repeat step ④ for washing; ⑦ Scan and detect fluorescence signals; (2) Data analysis: Normalize the raw data and screen for differentially inflammatory factors with logFC > 1.2 or < 0.83 and P < 0.05; (3) Syndrome classification: Based on the expression level of differential factors and bioinformatics analysis, the RA damp-heat obstruction syndrome and liver and kidney deficiency syndrome are distinguished.

5. The method according to claim 4, characterized in that, In step (1) ④, both washing solution I and washing solution II are diluted with deionized water to 20× concentrate. The shaking intensity is high during washing, and each shaking lasts for 10 seconds.

6. The method according to claim 4, characterized in that, In step (2), the differential factors were validated using the ELISA method, and the validation indicators included IL-6R, TIMP-2, and CXCL13.

7. The method according to claim 4, characterized in that, The differentially expressed inflammatory factors in step (2) include IL-6R, CXCL13, TIMP-2, TNF-RI, TNF-RII and PDGF-BB.

8. The method according to claim 4, characterized in that, The bioinformatics analysis in step (3) includes: GO enrichment analysis involves biological processes such as chemotactic regulation, TNF receptor activity, and extracellular matrix tissue. KEGG pathway analysis: involved JAK-STAT pathway, TNF pathway, and cytokine-receptor interaction pathway.

Images