Pathological detection method for atherosclerosis

Abstract

The invention discloses an atherosclerosis pathology detection method, and belongs to the technical field of pathology detection. The atherosclerosis pathology detection method is used for detecting instability factors of atherosclerosis plaques and comprises the following steps: performing proteolysis on a to-be-detected sample, and detecting whether the instability factors are enriched in proteolysis liquid or not; if the instability factor exists in the protein enzymatic hydrolysate, separating cells from the to-be-detected sample, carrying out single-cell RNA sequence analysis, and analyzing the proportion of endothelial cells in the separated cells; and constructing an endothelial cell model related to the instability factor, and determining whether the instability factor has a relationship with endothelial pyroptosis or not. According to the application, by detecting the instability factor, the relationship between the instability factor RIG-I and the atherosclerotic unstable plaque and pyroptosis is determined, and a new research thought is provided for prevention and treatment of atherosclerotic cardiovascular diseases.

Description

Technical Field

[0001] The present application relates to a method for detecting atherosclerosis pathology, and belongs to the technical field of pathology detection. Background Art

[0002] Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of death and disability in human diseases in China and even in the world. Atherosclerotic plaque formation is the most common pathological change in ASCVD. Studies have found that unstable plaques in the late stage of atherosclerosis (such as plaque rupture, thrombosis, and intraplaque hemorrhage) can lead to a series of acute clinical events, such as myocardial infarction and stroke. Therefore, the prevention and treatment of unstable atherosclerotic plaques is an important task in the prevention and treatment of cardiovascular diseases. However, existing treatment methods mostly start from strengthening the management of traditional risk factors and anti-oxidation and lipid-lowering, which can only play a delaying role and cannot achieve the purpose of accurately identifying unstable plaques and providing thorough treatment. Therefore, new therapeutic targets and more accurate diagnosis and treatment methods still need to be further explored.

[0003] The pathogenesis of atherosclerosis is relatively complex, involving functional changes in various cell types, such as endothelial cell dysfunction, inflammatory cell infiltration, and smooth muscle cell phenotypic transformation. Among them, functional changes in endothelial cells have multiple, mechanical effects on the initiation and progression of atherosclerotic plaques. In particular, the regenerative capacity impairment of endothelial cells is crucial for the reendothelialization of the surface-eroded arterial lumen and the stability of atherosclerotic plaques. It is worth noting that endothelial cell death is closely related to the regenerative capacity impairment of endothelial cells. On the one hand, endothelial cell death can not only directly cause thrombosis secondary to vascular denudation and cause acute cardiovascular events; on the other hand, it can also affect the surrounding environment through the damage-associated molecular patterns (DAMPs) of dead cells, aggravating the progression and instability of plaques. Therefore, continuous and intact endothelial cells are essential to inhibit the unstable progression of atherosclerotic plaques.

[0004] Cells in atherosclerotic lesions can die in a variety of ways, such as programmed cell death and necrosis. Among them, pyroptosis is a highly pro-inflammatory and lytic form of cell death. Studies on atherosclerotic plaques using transmission electron microscopy have shown that a large number of dead cells in plaques exhibit typical pyroptosis structural changes (such as membrane pore formation, cell lysis, etc.). After pyroptosis occurs, not only do the cells themselves rupture and dissolve, but more importantly, pyroptotic cells can promote thrombosis by promoting the production of tissue factor and the release of inflammatory factors, thereby inducing the most serious secondary lesions of atherosclerotic plaques. Therefore, pyroptosis is a key cell function change that drives the progression of plaque instability.

[0005] Retinoic acid-inducible gene-I (RIG-I, also known as DDX58) is a recently discovered important cytoplasmic sensor that plays a key role in innate immune response and inflammatory response. Many studies have found that RIG-I is associated with the occurrence of atherosclerosis. However, the specific effects of RIG-I on endothelial cells and its impact on the progression of plaque instability are still unclear. Therefore, in-depth exploration of the mechanism of action of RIG-I in the occurrence and development of atherosclerosis is expected to provide new theoretical basis for the therapeutic targets of atherosclerosis. Summary of the invention

[0006] The present application provides a method for detecting atherosclerosis pathology, which is used to detect the instability factors of atherosclerotic plaques, thereby providing a new theoretical basis for the treatment target of atherosclerosis.

[0007] The method for detecting atherosclerosis pathology comprises at least the following steps: Collect samples for testing; Performing proteolysis on the sample to be detected to obtain a proteolytic solution; and detecting whether the proteolytic solution is enriched with unstable factors; If the instability factor exists in the protease hydrolysate, separating cells from the sample to be tested, performing single-cell RNA sequence analysis, and analyzing the proportion of endothelial cells in the separated cells; An endothelial cell model associated with instability factors is constructed to confirm whether there is a relationship between the instability factors and endothelial cell pyroptosis.

[0008] Optionally, whether the protease hydrolysate is enriched with unstable factors is determined by performing chromatographic separation and mass spectrometry analysis on the protease hydrolysate.

[0009] Optionally, the method for obtaining the protease hydrolysate is: Lysing the sample to be detected to obtain a lysate; Extracting proteins from the lysate to obtain a protein sample; and performing quantitative and qualitative analysis on the protein sample; The protein sample is enzymatically hydrolyzed to obtain the protease hydrolyzate.

[0010] Optionally, the sample to be detected is lysed using SDT buffer.

[0011] Optionally, a BCA protein assay kit is used for quantitative analysis of protein; Cornell staining was used for qualitative analysis of proteins.

[0012] Optionally, an endothelial cell model associated with instability factors is constructed, specifically: an endothelial cell model with instability factor knockdown is constructed using lentivirus.

[0013] Optionally, constructing an endothelial cell model associated with an instability factor to confirm the relationship between the instability factor and endothelial cell pyroptosis includes: Healthy endothelial cells were infected with lentivirus to obtain an endothelial cell model associated with instability factors; The endothelial cell model is taken, treated with a cell pyroptosis stimulant to induce endothelial cell pyroptosis, and an experimental cell sample is obtained; Detecting the expression level of cell pyroptosis characteristic protein in the experimental cell sample, and judging whether there is a relationship between the instability factor and endothelial cell pyroptosis according to the expression level of the cell pyroptosis characteristic protein; Or detecting the release status of lactate dehydrogenase in the experimental cell sample, and judging whether the instability factor has a relationship with endothelial cell pyroptosis according to the release status of lactate dehydrogenase.

[0014] Optionally, the criterion for judging whether the instability factor has a relationship with endothelial cell pyroptosis according to the expression level of the cell pyroptosis characteristic protein is: if the expression level of the cell pyroptosis characteristic protein decreases, then there is a relationship between the instability factor and endothelial cell pyroptosis; The criterion for judging whether the instability factor has a relationship with endothelial cell pyroptosis according to the release of lactate dehydrogenase is: if the release of lactate dehydrogenase decreases, then there is a relationship between the instability factor and endothelial cell pyroptosis.

[0015] Optionally, the cell pyroptosis stimulator is oxidatively modified low-density lipoprotein ox-LDL.

[0016] Optionally, the instability factor is retinoic acid-inducible gene-I.

[0017] The beneficial effects of this application include: This application clarifies the relationship between the instability factor RIG-I and unstable atherosclerotic plaques and cell pyroptosis (especially endothelial cell pyroptosis) through the detection of instability factors, proposes new research ideas for the prevention and treatment of atherosclerotic cardiovascular diseases, and provides new clues for early molecular diagnostic markers and therapeutic targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Volcano plots of proteomic results for stable and unstable plaques; Figure 2 KEGG functional enrichment analysis histogram of the proteomic results of stable and unstable plaques; Figure 3 Cluster heatmap of proteomic results for stable and unstable plaques; Figure 4 This is a comparison of the mRNA expression levels of RIG-I in stable plaques and unstable plaques; Figure 5 This is a comparison of the mRNA expression levels of RIG-I in the peripheral blood of patients with stable plaques and patients with unstable plaques; Figure 6 single-cell atlas for plaque organization; Figure 7 The results of the effects of different concentrations of ox-LDL stimulation on the expression levels of different genes in endothelial cells are shown in Figure A, where Figure B is the expression level of RIG-I, Figure C is the expression level of CASP-1, and Figure C is the expression level of GSDMD; Figure 8 The results of the effects of different concentrations of ox-LDL stimulation on the level of LDH release in endothelial cells; Fig. 9 This is a comparison of the mRNA expression levels of CASP-1 in stable plaques and unstable plaque tissues; Fig.10 The histograms of the proportion of cells in stable and unstable plaques; Fig.11 The expression pattern of RIG-I in endothelial cells of total artery atherosclerotic plaque tissue; Fig.12 Western blot analysis of different pyroptosis genes and data analysis results (A is a Western blot; BD is the changes in the protein levels of RIG-I, caspase-1 (CASP-1), and GSDMD before and after simple RIG-I gene silencing; EG is the changes in the protein levels of RIG-I, CASP-1, and GSDMD before and after RIG-I gene silencing under ox-LDL stimulation); Fig.13 This is the result diagram of the effect of RIG-I gene silencing on the level of LDH release in endothelial cells; Fig.14 This figure shows the effect of RIG-I gene silencing on NLRP3 mRNA levels in endothelial cells. DETAILED DESCRIPTION

[0019] The present application is described in detail below with reference to the accompanying drawings and embodiments, but the present application is not limited to these embodiments.

[0020] Unless otherwise specified, the raw materials in the examples of this application were purchased through commercial channels.

[0021] The present application discloses a method for detecting atherosclerosis pathology, which is used to detect the instability factor RIG-I of atherosclerotic plaques; The method comprises at least the following steps: Step 1: Collect samples to be tested; Step 2, lysing the sample to be detected to obtain a lysate; Extracting protein from the lysate to obtain a protein sample; using a BCA protein detection kit and Kaunerst staining method to perform quantitative and qualitative analysis of the protein; Performing enzymatic hydrolysis on the protein sample to obtain the protease hydrolysate; The protease hydrolysate is subjected to chromatographic separation and mass spectrometry analysis to determine whether the protease hydrolysate is enriched with unstable factors.

[0022] The lysis of the sample to be tested is carried out by conventional methods in the art, including but not limited to crushing the tissue containing the lesion and adding a lysis reagent for lysis. The control of the specific operation process can be carried out by those skilled in the art according to the type of lysis reagent purchased or prepared. In the specific implementation process of this application, SDT buffer (composed of: 4% sodium dodecyl sulfate SDS aqueous solution by mass, 100mM tris-hydroxymethylaminomethane hydrochloride Tris-HCl, pH7.6) is used to perform preliminary lysis on the crushed tissue.

[0023] The protein detection kit used in the protein quantitative analysis of this application is the BCA (Bicinchoninic Acid) protein detection kit (Cat. No. P0012, produced by Shanghai Biyuntian).

[0024] Chromatographic separation and mass spectrometry analysis were performed using a nanoflow NanoElute system and a time-of-flight mass spectrometer (timsTOF Pro, Bruker, Germany) in PASEF mode. MS data were analyzed using protein spectrometry software (MaxQuant, version 1.6.17.0).

[0025] Step 3: If the protease hydrolysate contains the instability factor, cells are separated from the sample to be tested, single-cell RNA sequence analysis is performed, and the proportion of endothelial cells in the separated cells is analyzed; An endothelial cell model related to instability factors was constructed to confirm whether the instability factors were related to endothelial cell pyroptosis, specifically: Healthy endothelial cells were infected with lentivirus to obtain an endothelial cell model associated with instability factors; An endothelial cell model is taken, and treated with a cell pyroptosis stimulant to induce pyroptosis of the endothelial cells, thereby obtaining an experimental cell sample; Detecting the expression level of cell pyroptosis characteristic protein in the experimental cell sample, and judging whether there is a relationship between the instability factor and endothelial cell pyroptosis according to the expression level of the cell pyroptosis characteristic protein; Or detecting the release status of lactate dehydrogenase in the experimental cell sample, and judging whether the instability factor has a relationship with endothelial cell pyroptosis according to the release status of lactate dehydrogenase.

[0026] If the expression level of the cell pyroptosis characteristic protein is reduced, then there is a relationship between the instability factor and endothelial cell pyroptosis; If the release of lactate dehydrogenase (LDH) is reduced, there is a relationship between the instability factor and endothelial cell pyroptosis.

[0027] The methods for cell isolation and single-cell RNA sequence analysis from tissues are conventional technical means in the art. The actual operation process of this application includes: First, the tissue samples obtained after surgery are stored in MACS tissue storage solution (produced by Miltenyi-Biotec) before processing. The tissue samples are washed with phosphate-buffered saline (PBS) and cut into small pieces (about 1 mm) on ice. 3 ) and digested with 1 mg / ml collagenase I (Gibco), 1 mg / ml collagenase II, 60U / ml hyaluronidase II (Sigma-Aldrich), 10U / mL liberase (Roche) and 0.02mg / ml DNase I (Roche) at 37°C for 90 minutes. After digestion, the tissue samples were filtered through 100μm and 40μm meshes and centrifuged at 300g for 5 minutes. After removing the supernatant, the cell pellet was resuspended in red blood cell lysis buffer (Gibco), and the red blood cells were lysed and removed. After washing with 0.5% (mass volume concentration) bovine serum albumin BSA in Dulbecco's phosphate buffer D-PBS, the cells were resuspended in 0.5% (mass volume concentration) bovine serum albumin BSA in phosphate buffer PBS. Take out 5 μl of the single cell suspension solution, add 5 μl of trypan blue solution, mix, observe under a microscope, and count the cells with a cell counter to detect cell activity. Active cells above 85% are considered qualified. X Single Cell 3' Library Construction Kit (V3.1), based on 10 XThe official manual constructs a single-cell transcriptome 3' end sequencing library. The main experimental process includes: loading the cell suspension, single-cell 3' magnetic beads, and oil droplets onto the chip, and forming oil-in-water GEMs through the Chromium Controller microfluidics. In the oil-in-water, the cells rupture to release RNA, and the RNA is reverse transcribed to form cDNA with a labeled sequence barcode and construct a sequencing library. Finally, the single-cell cDNA library is sequenced.

[0028] During the implementation of this application, the sequencing work was assisted by Jikai Gene Biotechnology Co., Ltd. The sequencing data was quality-controlled using Cellranger 3.0 for the original sequencing files (GRCh38 was selected for the human reference genome); the data was quality-controlled using R language (4.2.2) in terms of the number of genes, the proportion of mitochondrial genes, and the expression level of ribosomal genes; the Runharmony function in the single-cell data integration software Harmony was used to integrate the data of different samples. The Uniform manifold approximation and projection (UMAP) dimensionality reduction method was used to visualize the single-cell population using the RunUMAP function and the FindClusters function.

[0029] For the single cell population results obtained, the type of each cell subpopulation was annotated based on the marker genes published in the existing literature, the cell marker database Cellmarker and the single cell database PanglaoDB, and then the proportion of endothelial cells among them was statistically calculated.

[0030] In order to ensure the health of endothelial cells and to avoid factors that cause cell pyroptosis, the healthy endothelial cells selected in this application are primary human umbilical vein endothelial cells (produced by Cybex Biotech). The experimental cell samples in this application are obtained as follows: Human primary umbilical vein endothelial cells were cultured in a basal medium containing primary endothelial cell culture additives and 10% (volume percentage) fetal bovine serum (including double antibodies) (produced by Cybio Biotech) and placed in an incubator at 37°C and 5% CO2. When the cell confluence reached about 80%, the cells were subcultured to maintain a good growth state.

[0031] Using lentivirus infection to obtain a cell model is a conventional technical means for those skilled in the art to obtain a cell model. This application uses a lentivirus infection method to obtain an endothelial cell model associated with an instability factor, specifically: Healthy cell samples, i.e. endothelial cells in the logarithmic growth phase, were inoculated into cell culture flasks, and 100 μl of culture medium (special culture medium for human umbilical vein endothelial cells, produced by Cebio) was added to each well. Incubate in a 37°C cell culture incubator overnight, and the infection was performed when the cell number reached 50% the next day. Gently mix the virus solution (based on HIV-I and using herpes virus VSVG capsid protein, produced by Shanghai Jikai Biology) with fresh complete culture medium and dilute it to the required concentration. Aspirate the original cell culture medium and add the diluted culture medium containing different virus solutions to the cells. Shake gently and place in a cell culture incubator overnight. After 16-24 hours of infection, replace with fresh culture medium, continue to culture the cells for 72 hours, and then replace with regular culture medium.

[0032] The cultured endothelial cell model was treated with ox-LDL for 12-20 hours to stimulate and induce endothelial cell pyroptosis to obtain an experimental cell sample.

[0033] In the present application, the expression level of the characteristic protein of cell pyroptosis is obtained by extracting the total protein of experimental endothelial cells related to the instability factor, and analyzing and calculating the total protein of the sample to be tested to obtain the relative expression amount of the protein.

[0034] In the present application, the lactate dehydrogenase release status is obtained by adding a lactate dehydrogenase releaser (produced by Beyotime) to the endothelial cells associated with the unstable factor and the human umbilical vein endothelial cells obtained by the above culture, and using an ELISA reader to detect the absorbance of each sample. That is, if the absorbance of the endothelial cell model sample associated with the unstable factor decreases, it indicates that there is a relationship between the unstable factor and endothelial cell pyroptosis.

[0035] The "sample to be tested" in this application refers to tissue containing atherosclerotic plaques. The samples used in the following verification experiments can be divided into stable plaque tissues and unstable plaque tissues.

[0036] 1. Sample collection Atherosclerotic plaques were obtained from patients who underwent carotid endarterectomy (CEA) in relevant hospitals in Inner Mongolia. All patients were diagnosed with carotid stenosis by CT angiography (CTA) before surgery, accompanied by clinical symptoms such as dizziness and cognitive dysfunction. The nature of plaques in patients with carotid stenosis was determined by preoperative imaging examinations and postoperative pathological analysis.

[0037] 2. Proteomics The preserved plaque tissue was taken and added with SDT buffer (4% sodium dodecyl sulfate SDS aqueous solution, 100mM tris-HCl, pH 7.6) to perform preliminary lysis of the tissue, and the extracted protein was quantified and quality tested using the BCA protein detection kit (P0012, Beyotime) and Kaunerst staining. The protein samples were enzymatically hydrolyzed, and chromatographic separation and mass spectrometry analysis were performed using the nanoliter flow rate NanoElute system and the PASEF mode of the time-of-flight mass spectrometer timsTOF Pro. The MS data were analyzed using MaxQuant software (version 1.6.17.0).

[0038] 3. Single-cell sequencing Fresh plaque tissue was used to prepare single-cell suspension by enzyme digestion, and trypan blue staining was used to evaluate the viability of the cell suspension. Emulsion gel beads were prepared from the cell suspension and single-cell cDNA library was constructed. Library sequencing was assisted by GeneCare Biotechnology Co., Ltd. The sequencing data were quality-controlled using Cellranger 3.0 for the original sequencing files (GRCh38 was selected as the human reference genome). The data were quality-controlled using R language (4.2.2) in terms of gene number, mitochondrial gene ratio, and ribosomal gene expression level. The Runharmony function in the single-cell data integration software Harmony was used to integrate data from different samples. The Uniform manifold approximation and projection (UMAP) dimensionality reduction method was used to visualize single-cell populations using the RunUMAP function and the FindClusters function. Type annotations were performed for each cell subpopulation based on marker genes reported in the literature and referenced to the Cellmarker database and the PanglaoDB database.

[0039] 4. Cell culture and lentiviral transfection Human primary umbilical vein endothelial cells were cultured in a basal medium containing primary endothelial cell culture additives and 10% (volume fraction) fetal bovine serum (including double antibodies) and placed in an incubator at 37°C and 5% CO2. When the cell confluence reached about 80%, the cells were subcultured to maintain a good growth state. The pyroptosis stimulator ox-LDL was treated for 16 hours to stimulate and induce endothelial cells. Before cell transfection, cells in the logarithmic growth phase were digested with trypsin, and the complete culture medium was used to make a cell suspension, and the corresponding number of cells was inoculated in the culture plate. Continue to culture, and replace the infection medium according to the results of the preliminary experiment, and add the optimal amount of virus for infection. About 8 hours after infection, replace the conventional culture medium (main components endothelial cell culture system (PriMed-iCell-002), fetal bovine serum (FBS), endothelial cell culture additives, double antibodies (penicillin / streptomycin, P / S)) and continue to culture.

[0040] 5. Western-blot Plaque tissue total protein extraction: Plaque tissues of patients undergoing endarterectomy were quickly frozen in liquid nitrogen, and then tissue protein lysis buffer was added. After lysis with a tissue disruptor, cells cultured in 6-well plates were taken, and total protein was extracted using tissue protein extraction reagent (produced by ThermoFisher), and protease inhibition mixture (produced by ThermoFisher) was added to inhibit protease, and protein concentration was determined by BCA method. After agarose gel electrophoresis, transfer, and blocking, primary antibody was added and incubated at 4°C for 2 hours, eluted, and secondary antibody was added and incubated at 4°C for 1 hour. Supersensitive luminescent solution (produced by Pierce™) was added for color development, and actin (abbreviated as β-Actin) was used as the internal reference protein to calculate the relative expression of protein.

[0041] 6. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) Plaque tissues of patients undergoing endarterectomy were quickly frozen in liquid nitrogen, and then added with tissue RNA lysis buffer. Total cellular RNA was extracted after lysis with the aid of a tissue disruptor. Peripheral blood was collected in EDTA anticoagulant tubes, and peripheral blood RNA was extracted according to the instructions of the Tiangen kit.

[0042] Total RNA extraction from cells: cells cultured in 6-well plates were taken and total RNA was extracted according to the instructions of the total RNA extraction kit (product name: RNAisoPlus, produced by Takara Bio-Tech Co., Ltd.). cDNA was prepared according to the instructions of Takara's reverse transcription kit, and genes were amplified by reverse transcription qPCR method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal reference gene. △△ The relative expression level of the target gene was calculated using the Ct formula.

[0043] 7. Lactate dehydrogenase release experiment Human umbilical vein endothelial cells growing in the logarithmic phase were taken, and the cell resuspension was added to a 96-well plate. When the cells grew to an appropriate density (70%-80%) under a microscope, the original culture medium was discarded and replaced with a low-serum culture medium. Ox-LDL stimulation was given according to the experimental groups, and the experimental groups were divided into a non-drug group and a drug-added group (50μg / ml). One hour before the end of the experiment, an LDH releaser (LDH release kit, produced by Shanghai Biyuntian) was added to the sample maximum enzyme activity control group (i.e., non-drug group), mixed, and continued to be placed in the incubator for culture. After reaching the time point, the 96-well plate was centrifuged first, and then the supernatant was taken from each well and placed in a new 96-well plate. An appropriate amount of LDH detection working liquid was added to each well and mixed. Incubate at room temperature in the dark, and detect the absorbance value of each well with an enzyme reader.

[0044] Verification experiment results analysis: This application conducts quantitative proteomics on stable and unstable plaques obtained from carotid endarterectomy.

[0045] The experimental results are as follows Figure 1-5 As shown, Figure 1-3 The following are the proteomics results of stable and unstable plaques. Figure 1 It is a volcano plot. The horizontal axis represents the logarithm of the difference in protein expression between the two samples, i.e., log2 (difference fold), and the vertical axis represents the statistical significance of the differential expression, which is the negative logarithm of the P value, i.e., -log10 (P value); following the principle of FC>1.2 or <0.83 and P value (t test) <0.05, blue dots represent down-regulated proteins in stable plaques, and red dots represent up-regulated proteins in unstable plaques (n=6 replicates / group). Figure 2 This is a histogram of KEGG (full name: Kyoto Encyclopedia of Genes and Genomes) functional enrichment analysis, showing the enriched signaling pathways between stable and unstable plaques (n=6 replicates / group). Figure 3 It is a cluster heat map, showing that the differential genes involved in immunity were significantly changed between stable plaques and unstable plaques (n=6 replicates / group) (the differential genes involved in immunity include three-motif protein 25 (TRIM25 for short), interferon gene stimulator 1 (STING1 for short), glyceraldehyde-3-phosphate dehydrogenase (TKFC for short), interferon stimulated gene 5 (ISG15 for short), TANK binding kinase 1 (TBK1 for short), human lung activation-regulated chemokine (CCL18 for short), β-2-microglobulin (B2M for short), and angiopoietin (ANG for short)). Figure 4This is a comparison of the relative expression levels of RIG-I in stable and unstable plaque tissues detected by qRT-PCR, i.e., the mRNA expression level of RIG-I in stable and unstable plaque tissues (**P < 0.01); Figure 5 This is a comparison chart of the relative expression of RIG-I in the peripheral blood of patients with stable plaques and unstable plaques detected by qRT-PCR, that is, the mRNA expression level of RIG-I in the peripheral blood of patients with stable plaques and unstable plaques (*P<0.05).

[0046] like Figure 1 As shown in the volcano plot, a total of 79 genes were significantly upregulated (n=32) or downregulated (n=37) compared with the stable patches. Figure 2 KEGG enrichment pathway analysis showed that several immune signaling pathways were significantly upregulated in unstable plaques, among which the RIG-I receptor signaling pathway was the most significantly enriched. Figure 3 As shown in the cluster heat map, multiple immune-related genes such as STING1 and TBK1 are also significantly upregulated in unstable plaques. Therefore, the activation of immune signaling pathways and the expression of related genes promote the progression of plaque instability. Given that most of the significantly upregulated immune genes are involved in the signal transduction of the RIG-I receptor signaling pathway, and RIG-I, as its upstream gene, regulates the expression of related immune genes, it can be seen that RIG-I is a new role involved in the progression of plaque stability.

[0047] To evaluate whether RIG-I is involved in the progression of plaque stability, this application used quantitative reverse transcription PCR (qRT-PCR experiment) for verification. Compared with the stable plaque population, the mRNA level of RIG-I in tissue and blood samples of the unstable plaque population was significantly increased (see Figure 4 , 5 ).

[0048] Atherosclerosis is characterized by heterogeneity and molecular complexity. In order to further analyze the composition and changes of endothelial cells in stable and unstable plaque tissues, and to clarify whether RIG-I is localized in atherosclerotic endothelial cells, this application isolated cells from stable and unstable plaque tissues and performed single-cell RNA sequence analysis. The analysis results are as follows Figure 6-10 As shown: in, Figure 6 Single-cell atlas of plaque tissue displayed by the dimensionality reduction algorithm UMAP; Figure 7 A shows the effect of different concentrations of ox-LDL on the expression level of RIG-I in endothelial cells (experimental data are expressed as mean ± standard error, *P < 0.05 vs 0 μg / ml); Figure 7B is the effect of different concentrations of ox-LDL on the expression level of caspase-1 (abbreviated as Caspase-1, or CASP-1) in endothelial cells (experimental data are expressed as mean ± standard error, *P < 0.05 vs 0 μg / ml); Figure 7 C shows the effect of different concentrations of ox-LDL on the expression level of membrane perforating protein Gasdermin D (GSDMD) in endothelial cells (experimental data are expressed as mean ± standard error, *P < 0.05 vs 0 μg / ml); Figure 8 The effect of different concentrations of ox-LDL on the level of LDH release in endothelial cells (experimental data are expressed as mean ± standard error, *P < 0.05 vs 0 μg / ml); Fig. 9 The results of qRT-PCR detection of the relative expression level of CASP-1 in stable and unstable plaque tissues (***P < 0.001); Fig.10 The histogram shows the proportion of cells in stable and unstable plaques (**P<0.01).

[0049] This analysis identified 13 different cell populations, including endothelial cells, based on the expression patterns of known cell marker genes (e.g. Figure 6 By calculating the proportion of endothelial cells in each sample, it was found that the proportion of endothelial cells in unstable plaques was significantly reduced compared with that in stable plaques (as shown in Figure 2). Fig.10 Thus, the reduction of endothelial cells may be the cause of the progression of atherosclerotic plaque instability.

[0050] Existing relevant studies have shown that the reduction of cells often indicates cell death, and microscopically, a large number of cells showed pyroptosis such as membrane damage and cell lysis during the unstable plaque stage. This suggests that endothelial cell pyroptosis may be an important functional change involved in the progression of plaque instability. To verify the above hypothesis, the pyroptosis stimulator ox-LDL was co-incubated with endothelial cells. Compared with the control, the expression levels of pyroptosis characteristic genes CASP-1 and GSDMD were significantly increased under ox-LDL stimulation (such as Figure 7 ), while the release of cellular LDH also increased significantly (as shown in Figure 8 Consistent with the in vitro studies, the mRNA level of CASP-1, a signature gene of pyroptosis, was significantly increased in unstable plaques compared with that in stable plaque tissues (as shown in Figure 2A). Fig. 9 as shown).

[0051] In order to further clarify whether RIG-I participates in the progression of plaque instability by inducing endothelial cell pyroptosis, this application used lentivirus to construct an endothelial cell model with RIG-I knockdown (i.e., gene silencing) and conducted a verification experiment. Figure 11-14 As shown below: Fig.11 The expression pattern of RIG-I (or DDX58) in endothelial cells of total atherosclerotic plaque tissue indicates that RIG-I is a gene stably expressed in endothelial cells in the above single-cell sequencing results. Therefore, this study used lentivirus to construct an endothelial cell model with RIG-I knockdown to clarify whether RIG-I participates in the progression of plaque instability by inducing endothelial cell pyroptosis.

[0052] Fig.12 The following are the Western blot analysis and data analysis results of the characteristic genes of pyroptosis in different cells. Fig.12 A: Western blot analysis, where the horizontal axis represents the groups (Control represents normal endothelial cells; Con+ox-LDL represents endothelial cells + oxidized low-density lipoprotein, normal endothelial cells treated with ox-LDL; RIG-I - / - Indicates RIG-I gene silencing, RIG-I - / - +ox-LDL indicates RIG-I gene silencing + oxidized low-density lipoprotein, i.e., RIG-I knockdown followed by ox-LDL treatment); the vertical axis indicates the expression levels of genes for Western blotting (i.e., RIG-I, CASP-1, GSDMD). Fig.12 BD: Effects of knockdown of RIG-I alone on changes in RIG-I, CASP-1, and GSDMD protein levels. Fig.12 EG: Effects of RIG-I knockdown on the changes in RIG-I, CASP-1, and GSDMD protein levels before and after ox-LDL stimulation (n = 3 independent experiments; *P<0.05**P<0.01); Fig.13 The effect of RIG-I knockdown on the level of LDH release in endothelial cells after ox-LDL stimulation (experimental data are expressed as mean ± standard error, *P < 0.05 vs 0ug / ml); Fig.14 The effect of RIG-I knockdown on NLRP3 mRNA levels in endothelial cells (n = 3 independent experiments; **P < 0.01).

[0053] It can be seen that compared with the control normal endothelial cells, RIG-I knockdown significantly reduced the expression level of cell pyroptosis characteristic proteins under ox-LDL stimulation, and at the same time, LDH release was also significantly reduced. But interestingly, in the above research results, RIG-I knockdown does not seem to participate in the occurrence of cell pyroptosis by affecting the expression of the traditional inflammasome molecule Nod-like receptor protein 3 (NLRP3 for short).

[0054] As a pro-inflammatory form of cell death, pyroptosis plays an important role in the initiation, progression and complications of atherosclerosis. However, the specific molecular mechanism by which pyroptosis causes plaque instability has not yet been elucidated. This application verifies through the above experiments that RIG-I is involved in inducing the occurrence of pyroptosis, and is expected to propose a new mechanism for the prevention and treatment of atherosclerosis-related diseases and the inhibition of plaque instability.

Claims

1. A method for detecting atherosclerosis pathology, characterized in that: The method is used to detect the instability factor of atherosclerotic plaque; The method comprises at least the following steps: Collect samples for testing; Performing proteolysis on the sample to be detected to obtain a proteolytic solution; and detecting whether the proteolytic solution is enriched with unstable factors; If the instability factor exists in the protease hydrolysate, separating cells from the sample to be tested, performing single-cell RNA sequence analysis, and analyzing the proportion of endothelial cells in the separated cells; An endothelial cell model associated with instability factors is constructed to confirm whether there is a relationship between the instability factors and endothelial cell pyroptosis.

2. The method for detecting atherosclerosis pathology according to claim 1, characterized in that: Whether the protease hydrolysate is enriched with unstable factors can be determined by performing chromatographic separation and mass spectrometry analysis on the protease hydrolysate.

3. The method for detecting atherosclerosis pathology according to claim 1, characterized in that: The method for obtaining the protease hydrolysate is as follows: Lysing the sample to be detected to obtain a lysate; Extracting proteins from the lysate to obtain a protein sample; and performing quantitative and qualitative analysis on the protein sample; The protein sample is enzymatically hydrolyzed to obtain the protease hydrolyzate.

4. The method for detecting atherosclerosis pathology according to claim 3, characterized in that: The sample to be detected is lysed using SDT buffer.

5. The method for detecting atherosclerosis pathology according to claim 3, characterized in that: BCA protein detection kit was used for quantitative analysis of protein; Cornell staining was used for qualitative analysis of proteins.

6. The method for detecting atherosclerosis pathology according to claim 1, characterized in that: Construct an endothelial cell model associated with instability factors, specifically: use lentivirus to construct an endothelial cell model with instability factor knockdown.

7. The method for detecting atherosclerosis pathology according to claim 1 or 6, characterized in that: Constructing an endothelial cell model associated with an instability factor and confirming the relationship between the instability factor and endothelial cell pyroptosis, including: Healthy endothelial cells were infected with lentivirus to obtain an endothelial cell model associated with instability factors; The endothelial cell model is taken, treated with a cell pyroptosis stimulant to induce endothelial cell pyroptosis, and an experimental cell sample is obtained; Detecting the expression level of cell pyroptosis characteristic protein in the experimental cell sample, and judging whether there is a relationship between the instability factor and endothelial cell pyroptosis according to the expression level of the cell pyroptosis characteristic protein; Or detecting the release status of lactate dehydrogenase in the experimental cell sample, and judging whether the instability factor has a relationship with endothelial cell pyroptosis according to the release status of lactate dehydrogenase.

8. The method for detecting atherosclerosis pathology according to claim 7, characterized in that: The criterion for judging whether the instability factor has a relationship with endothelial cell pyroptosis according to the expression level of the cell pyroptosis characteristic protein is: if the expression level of the cell pyroptosis characteristic protein decreases, then there is a relationship between the instability factor and endothelial cell pyroptosis; The criterion for judging whether the instability factor has a relationship with endothelial cell pyroptosis according to the release of lactate dehydrogenase is: if the release of lactate dehydrogenase decreases, then there is a relationship between the instability factor and endothelial cell pyroptosis.

9. The method for detecting atherosclerosis pathology according to claim 7, characterized in that: The cell pyroptosis stimulant is oxidatively modified low-density lipoprotein ox-LDL.

10. The method for detecting atherosclerosis pathology according to any one of claims 1 to 6, characterized in that: The instability factor is retinoic acid-inducible gene-I.

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