Application of secretory protein CTSB in predicting the risk of peripheral vascular disease in patients with DFU

Abstract

The present invention provides the application of the secretory protein CTSB (cathepsin B) in predicting the risk of vascular lesions in patients with diabetic foot ulcers (DFUs), belonging to the field of biomedicine technology. Through single-cell sequencing studies, the present invention found that the secretory protein CTSB is significantly enriched in vascular endothelial cells in the blood vessels of DFU patients, with high PPI connectivity and low expression; CTSB overexpression / knockout experiments found that CTSB is associated with the proliferation and tubulogenesis of vascular endothelial cells in human blood vessels; ELISA experiments also found that the level of secretory protein CTSB in serum can be used as a marker for predicting the risk of vascular lesions in DFU patients, and that the expression level of CTSB is negatively correlated with the Wanger grade of diabetic foot; the method can predict the risk of peripheral vascular lesions in DFU patients, has the advantages of simple operation and high sensitivity, can reduce false detection and missed detection, and has stable and reliable test results, making it suitable for large-scale detection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the application of the secretory protein CTSB in serum in predicting the risk of developing vascular lesions in patients with dilated fistula (DFU). Background Art

[0002] Diabetic foot ulcers (DFUs) are a common chronic complication of diabetes mellitus (DM) and a leading cause of disability and mortality in DM patients. Globally, 4%-10% of DM patients suffer from DFUs, with a lifetime morbidity of 25%, an annual mortality rate of 11%, and a high rate of 22% for amputees, with a 5-year mortality rate exceeding 70%. Furthermore, DFUs take a median of 32 weeks to heal, presenting a significant public health problem that places a heavy burden on society. Peripheral vascular disease, peripheral neuropathy, and foot deformities are the primary causes of DFUs, with 50% of DFU patients suffering from peripheral vascular disease. The destruction of peripheral vessels, reduced neovascularization, impaired revascularization, and vascular dysfunction in peripheral vascular disease are key factors contributing to the difficulty in healing diabetic ulcers. Early detection, diagnosis, and treatment can effectively improve DFU outcomes. However, approximately 50% of DFU patients often miss the optimal treatment window due to the absence of typical manifestations. Furthermore, the frequent presence of arterial calcification and constriction in diabetic patients makes DFU diagnosis challenging, leading to the development of chronic ulcers that fail to heal.

[0003] Currently, diagnostic methods for peripheral vascular disease in DFUs include the ankle-brachial index, toe-brachial index, transcutaneous oxygen tension, Doppler ultrasound, magnetic resonance angiography, and digital subtraction angiography. However, these diagnostic methods for peripheral vascular disease in DFUs have several limitations: First, they are all non-invasive screening tests with insufficient sensitivity and specificity, and cannot completely and reliably exclude DFU peripheral vascular disease. Furthermore, the revascularization time of DFUs is short, and these diagnostic methods have a diagnostic lag. These limitations mean that there is an urgent need for sensitive, specific, comprehensive, and rapid diagnostic markers to assess the risk of peripheral vascular disease in patients with DFUs. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide the application of the secretory protein CTSB in predicting the risk of vascular lesions in patients with diabetic foot disease. Through single-cell sequencing studies, the present invention found that the secretory protein CTSB is significantly enriched in the vascular endothelial cells in the blood vessels of patients with DFU, with high PPI connectivity and low expression; CTSB overexpression / knockout experiments found that CTSB is associated with the proliferation and tubulogenesis of vascular endothelial cells in human blood vessels under a high-glucose environment, indicating that CTSB is associated with the vascular lesions of DFU; ELISA experiments also found that the level of secretory protein CTSB in serum can be used as a marker for predicting the risk of vascular lesions in patients with DFU, and the expression level of CTSB is negatively correlated with the Wanger grade of diabetic foot. Therefore, the secretory protein CTSB in serum can be used as a marker for predicting the risk of vascular lesions in patients with DFU.

[0005] The present invention provides the use of a reagent for detecting the content of the secretory protein CTSB (i.e., cathepsin B) in the preparation of a reagent or kit for predicting the risk of vascular lesions in DFU patients. When the content of the secretory protein CTSB is significantly lower than that of healthy individuals, it is determined that the individual to be tested has a risk of vascular lesions.

[0006] Preferably, the secretory protein CTSB is the secretory protein CTSB in peripheral blood; the sample used to detect the secretory protein CTSB is preferably peripheral blood, serum or plasma.

[0007] Preferably, the reagent for detecting the content of secretory protein CTSB includes an ELISA detection reagent.

[0008] Preferably, overexpression of the secretory protein CTSB in a high glucose environment promotes the proliferation and tubule formation of vascular endothelial cells in the blood vessels of the DFU wound.

[0009] Preferably, knocking down the secretory protein CTSB in a high glucose environment inhibits the proliferation and tubule formation of vascular endothelial cells in the blood vessels of the DFU wound.

[0010] Compared with the existing technology, the present invention has the following beneficial effects: the present invention provides a reagent for detecting the content of secretory protein CTSB (cathepsin B) for use in preparing a reagent or kit for predicting the risk of vascular lesions in DFU patients. When the content of secretory protein CTSB is significantly lower than that of healthy individuals, it is determined that the individual to be tested has a risk of vascular lesions.

[0011] The present invention, through single-cell sequencing, found that the secreted protein CTSB is significantly enriched in vascular endothelial cells in the blood vessels of patients with diabetic foot (DFU), with high PPI connectivity and low expression. CTSB overexpression / knockout experiments found that CTSB is associated with the proliferation and tubulogenesis of vascular endothelial cells in human blood vessels. ELISA experiments also found that serum secreted protein CTSB levels can serve as a marker for predicting the risk of vascular lesions in patients with DFU, and that CTSB expression levels are negatively correlated with the Wanger grade of diabetic foot. Therefore, serum secreted protein CTSB is used as a marker for predicting the risk of peripheral vascular lesions in patients with DFU. This invention provides a new method for predicting the risk of peripheral vascular lesions in patients with DFU, which has important clinical value.

[0012] The method provided by the present invention is helpful to predict the risk of peripheral vascular disease in DFU patients at an early stage. The present invention has the advantages of simple operation and high sensitivity, is easy to use, and can reduce false detection and missed detection.

[0013] The present invention adopts the ELISA method to detect the content of secretory protein CTSB in peripheral blood. The method is simple to use, short in time, low in cost, and the detection results are stable and reliable, and is suitable for large-scale detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the gene network connection diagram of the single-cell transcriptome PPI combined with the EcCentricity algorithm;

[0015] Figure 2 This is a diagram of cell proliferation analysis of the siNC group, si-CTSB group, oeNC group, and oe-CTSB group in Example 2;

[0016] Figure 3 This is a diagram showing analysis of cell tubule formation in the siNC group, si-CTSB group, oeNC group, and oe-CTSB group in Example 3;

[0017] Figure 4 This is an analysis chart of CTSB expression levels in the normal group, DFU group, and DFUs of different Wanger grades detected by ELISA. DETAILED DESCRIPTION

[0018] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0019] Example 1

[0020] Mining genes or their expression products related to DFU vasculopathy by single-cell sequencing

[0021] The implementation steps are as follows:

[0022] Single-cell sequencing

[0023] The single-cell transcriptome gene expression datasets GSE165816 and GSE223964 (containing foot ulcer skin samples from 9 DFU patients and 4 normal foot skin samples) were downloaded from the GEO database. KEGG enrichment analysis of single-cell transcriptomes was performed using the clusterProfiler software package to identify genes significantly enriched in the vascular endothelial cells of DFU. It was found that the expression of CTSB in DFU patients was significantly lower than that in normal skin, and the gene was specifically expressed in vascular endothelial cells. The enriched gene connection network obtained by combining the PPI of single-cell transcriptomes with the EcCentricity algorithm was found to have a high PPI connectivity in the gene network enriched in DFU and is a secreted protein (see Figure 1 ).

[0024] Table 1 CTSB gene enrichment analysis results

[0025]

[0026] Example 2

[0027] CCK8 assay verifies that CTSB expression is associated with the proliferation of vascular endothelial cells in DFU wound vessels

[0028] The implementation steps are as follows:

[0029] Experimental groups:

[0030] si-CTSB group: CTSB gene was knocked down in vascular endothelial cells. The interference sequence was purchased from Suzhou Genema Gene Co., Ltd.;

[0031] siNC group: control group for knocking down the target gene in vascular endothelial cells. The interference sequence was purchased from Suzhou Genema Gene Co., Ltd.;

[0032] oe-CTSB: CTSB gene was overexpressed in vascular endothelial cells. This expression vector was purchased from Guangzhou Dongze Biotechnology Co., Ltd.

[0033] oeNC group: control group with overexpression of target gene in vascular endothelial cells. The expression vector was purchased from Guangzhou Dongze Biotechnology Co., Ltd.

[0034] Endothelial cells transduced with siNC, si-CTSB, oeNC, and oe-CTSB were seeded at 5,000 cells per well in 96-well plates, and 100 μL of high-glucose medium was added to each well. The plates were cultured in a 37°C, 5% CO2 incubator. The plates were removed at 0, 24, 48, 72, and 96 hours of incubation, and 10 μL of CCK8 solution (5 mg / mL) was added per well. After incubation for 2 hours, the absorbance (OD) of each well was measured at 450 nm. A higher OD value indicates a higher number of viable cells and higher cell viability. A CCK8 curve was plotted, with the interval time plotted on the horizontal axis and the absorbance on the vertical axis. The experiments revealed that under high-glucose conditions, cell proliferation in the si-CTSB group was significantly slower than that in the siNC group, confirming that CTSB knockdown inhibits the proliferation of endothelial cells in DFU wounds. Through experiments, it was found that the cell proliferation rate of the oe-CTSB group was significantly faster than that of the oeNC group under high glucose conditions, which confirmed that the overexpression of CTSB can promote the proliferation of vascular endothelial cells in DFU wound vessels (see Figure 2 ).

[0035] Example 3

[0036] Tube formation experiments verified that CTSB expression was associated with tube formation in vascular endothelial cells in DFU wound vessels

[0037] The implementation steps are as follows:

[0038] Matrigel was added to a 24-well plate at a volume of 50 μL per well, spread evenly and placed at 4°C overnight. The 24-well plate was then placed in a 37°C, 5% CO2 incubator for 1 hour to allow the matrigel to solidify. The vascular endothelial cells transduced by the siNC group, si-CTSB group, oeNC group, and oe-CTSB group were seeded in a 24-well plate at 150,000 cells per well, and 500 μL of high-glucose culture medium was added to each well. The cells were incubated at 37°C for 24 hours, and the tube formation was observed under a confocal microscope and photographed. Through experiments, it was found that the cell proliferation rate of the si-CTSB group was significantly slower than that of the siNC group under a high-glucose environment, which confirmed that the knockdown of CTSB can inhibit the tubule formation of vascular endothelial cells in the DFU wound blood vessels. Through experiments, it was found that the cell proliferation rate of the oe-CTSB group was significantly faster than that of the oeNC group under a high-glucose environment, which confirmed that the overexpression of CTSB can promote the tubule formation of vascular endothelial cells in the DFU wound blood vessels (see Figure 3 ).

[0039] Example 4

[0040] Enzyme-linked immunosorbent assay was used to detect the expression of CTSB in patients

[0041] Specific testing process:

[0042] CTSB antigen was dissolved in 50 mM carbonate coating buffer (pH 9.6) to a concentration of 15 μg / mL. 100 μL / well of the plate was added to a 96-well ELISA plate and incubated at 4°C overnight. The coating buffer was discarded the next day and the plate was washed three times with PBST. 5% BSA was added to block the plate at 37°C for 40 minutes. During blocking, the blocking buffer was filled to the top of each well and air bubbles were removed. After blocking, the plate was washed three times with PBST. The above steps were repeated, and the blocked plate was divided into three groups, each containing 17 wells. Diluted serum samples from the healthy subjects and the DFU group were tested by ELISA. Serum samples from the DFU group with different Wanger grades (Wanger 3, Wanger 4, and Wanger 5) were added to the ELISA reaction wells at 100 μL per well. The plates were incubated at 37°C for 40 minutes and washed three times with PBST. Add enzyme-labeled antibody, 100 μL per well, incubate at 37°C for 60 minutes, and wash three times with PBST. Add TMB-urea hydrogen peroxide solution as substrate solution, 100 μL per well, incubate at 37°C in the dark for 10 minutes. Add 50 μL of terminator to terminate the reaction and measure the experimental results. Use a microplate reader to read the data, with a wavelength of 450 nm, and calculate the corresponding concentration based on the OD value. Through experiments, it was found that the expression level of CTSB in the DFU patient group was lower than that in the healthy group, and its expression level decreased with the increase of DFU Wanger grade, which further proved that the expression level of CTSB was negatively correlated with the risk of vascular lesions in DFU (see Figure 4 ).

[0043] Table 2 OD value test results of serum samples from healthy group and DFU group

[0044]

[0045] Table 3 OD value test results of serum samples in the DFU group with different Wanger grades

[0046]

[0047] The results of the above examples show that the secreted protein CTSB is significantly enriched and lowly expressed in vascular endothelial cells in DFU wounds. The ELSIA expression level of this secreted protein is negatively correlated with the Wanger grade of DFU. The mechanism by which CTSB promotes the pathogenesis of DFU is related to its inhibition of the proliferation and tubule formation of vascular endothelial cells in the patient's wound. These results suggest that the secreted protein CTSB can be used as a marker to predict the risk of DFU peripheral vascular disease.

[0048] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of a reagent for detecting the content of secretory protein CTSB in a reagent or kit for predicting the risk of vascular lesions in patients with DFU, characterized in that: When the content of the secretory protein CTSB in the serum of a DFU patient is significantly lower than the average CTSB content of a healthy person, it is determined that the DFU patient to be tested has a risk of developing vascular lesions.

2. The use according to claim 1, characterized in that The secretory protein CTSB is the secretory protein CTSB in peripheral blood.

3. The use according to claim 1 or 2, characterized in that Reagents for detecting the content of secretory protein CTSB include ELISA detection reagents.

4. The use according to claim 1, characterized in that Overexpression of the secretory protein CTSB in a high glucose environment promotes the proliferation and tubule formation of vascular endothelial cells in DFU wound vessels.

5. The use according to claim 1, characterized in that Knockdown of the secretory protein CTSB in a high-glucose environment inhibits the proliferation and tubule formation of vascular endothelial cells in DFU wound vessels.

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