Application of a biomarker GSDME

Abstract

The present invention discloses an application of a biomarker GSDME. The application includes the use of the biomarker GSDME protein in preparing a kit for diagnosing osteoarthritis OA subtypes, wherein the kit contains a detection reagent and a detection tool for detecting the expression level of the biomarker GSDME protein, and the kit is also used for prognosis assessment of osteoarthritis OA subtypes. The present invention is based on the GSDME protein with high specificity, versatility and reliability as a biomarker, and prepares a kit that can more simply and conveniently detect the GSDME protein, which is easy to use and easy to promote and popularize.

Description

Technical Field

[0001] The present invention relates to an application of a biomarker GSDME, and in particular to an application of the biomarker GSDME in preparing a diagnostic kit for osteoarthritis subtypes. Background Art

[0002] Osteoarthritis (OA) is a common joint degenerative disease in the elderly. It affects the synovium and cartilage, leading to joint destruction and a series of clinical symptoms. It is currently believed that the pathological process of osteoarthritis includes progressive loss and destruction of cartilage, subchondral bone sclerosis, osteophyte formation, varying degrees of synovial inflammation, ligament and meniscus degeneration, etc. The clinical manifestations are joint pain, short-term morning stiffness, and movement disorders. When it develops to the end stage, joint replacement is necessary. This also makes knee osteoarthritis one of the main causes of disability in middle-aged and elderly people. At present, due to the lack of clarity about the pathological mechanism of osteoarthritis, there has been no breakthrough in the diagnosis and treatment of early osteoarthritis. The commonly used diagnostic methods for osteoarthritis can be roughly divided into two categories. One is diagnosis with the help of images such as X-ray, ultrasound and MRI, and the other is diagnosis through osteoarthritis-related biomarkers. Currently, biomarkers that can be used to diagnose osteoarthritis include type II collagen C-terminal peptide, matrix metalloproteinases, tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1). However, since most biomarkers are driven by a variety of pathophysiological factors and are also affected by drug interventions, resulting in inaccurate measurements, and some are unable to effectively predict the severity and progression of arthritis, new and reliable biomarkers are urgently needed to achieve reliable diagnosis of osteoarthritis.

[0003] In addition, the current detection of biomarkers in osteoarthritis is often carried out through qPCR, tissue section immunohistochemistry, etc. The detection process has many steps, is time-consuming, and needs to be carried out in a laboratory with professional conditions. Therefore, it cannot be achieved in some grassroots hospitals that are not equipped with laboratory conditions. Therefore, a convenient and reliable method for diagnosing osteoarthritis is urgently needed.

[0004] Apoptosis and autophagy are two major mechanisms of osteoarthritis, while pyroptosis is a type of programmed inflammatory cell death that is different from them. It is characterized by continuous cell swelling until the cell membrane ruptures, resulting in the release of cell contents and causing a strong inflammatory response. Recently, researchers have made new breakthroughs in the field of pyroptosis, revealing the mechanism of GSDME (gasdermin E) in the gasdermin family mediating pyroptosis. This protein is mainly activated by caspase3 or caspase8 and can induce the transition of non-inflammatory cell apoptosis to pyroptosis. At present, there have been related studies on GSDME in osteoarthritis, revealing the correlation between osteoarthritis and GSDME expression levels. Therefore, GSDME has great potential as a diagnostic biomarker for osteoarthritis. Summary of the invention

[0005] In order to solve the problems existing in the background technology, the purpose of the present invention is to provide a biomarker GSDME for use in the preparation of an osteoarthritis subtype diagnostic kit for the diagnosis and prognosis of osteoarthritis. In order to achieve the above purpose and other related purposes, the present invention proves at the cell, animal and human levels that the GSDME protein increases significantly after osteoarthritis. Therefore, osteoarthritis can be diagnosed and predicted by detecting the level of GSDME protein in the joint fluid of the subject, providing a new approach for the diagnosis of arthritis. The first aspect of the present invention provides the use of GSDME protein as a biomarker in a detection kit for detecting the severity and prognosis of osteoarthritis and / or evaluating the severity of osteoarthritis and the efficacy of the disease. The second aspect of the present invention provides a diagnostic kit for detecting osteoarthritis subtypes.

[0006] The technical solution adopted by the present invention is:

[0007] 1. Application of biomarker GSDME

[0008] The biomarker GSDME protein is used in preparing a kit for diagnosing osteoarthritis OA subtypes.

[0009] The biomarker GSDME protein is gasdermin E protein.

[0010] The kit contains detection reagents and detection tools for detecting the expression level of the biomarker GSDME protein. The detection reagents include: human GSDME protein standard, anti-GSDME antibody, rabbit anti-human IgG H&G antibody, color reaction substrate solution and reaction termination solution; the detection tools include: ELISA plate.

[0011] The human GSDME protein standard is specifically recombinant human GSDME protein;

[0012] The color development reaction substrate solution is specifically a tetramethylbenzidine solution;

[0013] The reaction termination solution is specifically a 2M sulfuric acid solution.

[0014] The kit is also used for prognosis assessment of osteoarthritis OA subtypes. The expression level of GSDME protein is negatively correlated with the prognosis of osteoarthritis OA subtypes.

[0015] 2. A method for diagnosing osteoarthritis subtypes

[0016] The method includes: obtaining a sample solution, and detecting whether the expression level of a biomarker GSDME protein in the sample solution is abnormally increased. If an abnormal increase is found, it indicates osteoarthritis belonging to the osteoarthritis OA subtype.

[0017] The biomarker GSDME protein is gasdermin E protein; the sample solution is specifically joint fluid.

[0018] The expression level of the biomarker GSDME protein was quantitatively detected using enzyme-linked immunosorbent assay (ELISA).

[0019] The biomarker GSDME protein is derived from an in vitro solution, for example, it can be derived from a non-living tissue fluid.

[0020] The beneficial effects of the present invention are:

[0021] The present invention provides a biomarker GSDME for preparing a diagnostic kit for osteoarthritis subtypes, and establishes a practical, sensitive and specific immunological detection method for GSDME in synovial fluid. The research of the present invention shows that GSDME protein is a biomarker with high specificity, versatility and reliability, which is easy to popularize and popularize for predicting the severity and prognosis evaluation of osteoarthritis, and can provide an objective basis for judging the prognosis of osteoarthritis patients and formulating a reasonable treatment plan; moreover, compared with obtaining cartilage tissue samples by biopsy to detect the expression of GSDME protein, synovial fluid samples are easier to obtain, which is more convenient for detecting the expression level of GSDME protein, and the detection method of GSDME protein is simpler and easier to promote and apply in clinical or primary hospitals; in addition, the GSDME protein immunoassay method established by the present invention provides a reliable biological diagnosis basis for osteoarthritis, which helps the current diagnosis method based on clinical data. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The present invention collects the analysis results of RNA-seq technology of transcriptome sequencing of synovial fluid of nearly 100 OA patients and other patients, as well as a schematic diagram of clinical data based on GSDME protein classification;

[0023] Figure 2 It is a schematic diagram of the immunofluorescence results of the expression level of GSDME protein in the knee joint tissue samples of the operation group and the sham operation group in the animal experiment of the present invention;

[0024] Figure 3 It is a schematic diagram of detecting the expression level of GSDME protein in cartilage tissue of the surgical group by Western Blotting (WB) in the animal experiment of the present invention;

[0025] Figure 4 This is a schematic diagram of the results of two groups of randomized comparative experiments using the enzyme-linked immunosorbent assay (ELISA) method to detect the expression levels of GSDME protein in synovial fluid samples of osteoarthritis subjects and non-osteoarthritis subjects in the main experiment of the present invention. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0027] Example:

[0028] Human experiments were conducted, and synovial fluid from nearly 100 patients with osteoarthritis OA subtype and other patients in a hospital and synovial fluid from several non-living animals collected in Example 2 were collected. The inclusion criteria for the above-mentioned osteoarthritis OA subtype patients were: age ≥ 18 years old, and at the same time met a) diagnosed with knee osteoarthritis according to internationally recognized diagnostic criteria; 2) K / L score of 2 to 3; 3) chronic knee joint pain; b) no local or systemic infection; c) no obvious contraindications to joint puncture in hematological and biochemical tests; d) 0.5 ml of joint effusion was extracted from the patient. If joint effusion could not be extracted, the patient was not included in the group; the exclusion criteria were a) age greater than 75 years or less than 18 years old; b) HIV, hepatitis virus or syphilis virus infection or positive serological test; c) body mass index (BMI) greater than 30 kg / m 2 ; d) congenital or acquired knee deformity; e) pregnant or lactating women; f) tumor patients; g) immunodeficiency patients; h) patients currently participating in other clinical trials; i) patients considered unsuitable for inclusion by other researchers. This study was conducted in accordance with the principles of medical ethics.

[0029] The synovial fluid of patients with osteoarthritis subtype OA and the synovial fluid of other patients are collectively referred to as control samples in the following steps, and the expression level of GSDME protein in the control samples is detected by ELISA:

[0030] 1) Prepare a kit required for enzyme-linked immunosorbent assay (ELISA), wherein the kit contains detection reagents and detection tools for detecting the expression level of GSDME protein, wherein the detection reagents include: human GSDME protein standard, anti-GSDME antibody, rabbit anti-human IgG H&G antibody, color reaction substrate solution and reaction termination solution; the detection tools include: ELISA plate; the human GSDME protein standard is specifically recombinant human GSDME protein; the color reaction substrate solution is specifically tetramethylbenzidine solution (TMB); the reaction termination solution is specifically 2M sulfuric acid solution.

[0031] 2) Take the anti-GSDME antibody in the kit and use it to coat each well on the ELISA plate; add 100 μL of control sample or recombinant human GSDME protein to each well, incubate at 37°C for 30 minutes, and repeat aspiration / rinsing of each well 3 times.

[0032] 3) Take the rabbit anti-human IgG H&G antibody in the kit, dilute the rabbit anti-human IgG H&G antibody to a volume ratio of 1:5000-1:200000, add 100 μL of the diluted rabbit anti-human IgG H&G antibody to each well of the ELISA plate, incubate at 37°C for 30 minutes, and repeat aspiration / rinsing of each well 3 times.

[0033] 4) Take the tetramethylbenzidine solution from the kit, add 100 μL of the tetramethylbenzidine solution to each well of the ELISA plate, and incubate at room temperature for 30 minutes.

[0034] 5) Take a 2M sulfuric acid solution from the kit and add 50 μL of the 2M sulfuric acid solution to each well of the ELISA plate.

[0035] 6) Use a microplate reader to measure the absorbance of the ELISA plate at a maximum absorption wavelength of 450 nm.

[0036] For the recombinant human GSDME protein, the protein concentration of the recombinant human GSDME protein is known, and a linear regression fitting method is used to construct an absorbance-GSDME protein concentration standard curve between the absorbance measured using the recombinant human GSDME protein in the above step and the protein concentration of the recombinant human GSDME protein.

[0037] For the control sample, the absorbance is measured using the control sample in the above steps, and the absorbance measured by the control sample is substituted into the absorbance-GSDME protein concentration standard curve to obtain the GSDME protein concentration of the control sample, that is, the expression level of the GSDME protein concentration in the synovial fluid of patients with osteoarthritis OA subtype and other patients is obtained.

[0038] like Figure 4As shown, it is a schematic diagram of the comparison of two randomly selected comparison groups. The left figure shows the comparison between 2 other patients and 5 osteoarthritis OA subtype patients, and the right figure shows the comparison between 4 other patients and 16 osteoarthritis OA subtype patients. The results show that the expression level of GSDME protein in the synovial fluid of osteoarthritis OA subtype patients is higher than that of GSDME protein in the synovial fluid of other patients, and in osteoarthritis OA subtype patients, the expression level of GSDME protein increases with the severity of arthritis, and with the increase of the severity of arthritis, the expression level of GSDME protein in the synovial fluid of osteoarthritis OA subtype patients is about 2-6 times the expression level of GSDME protein in the synovial fluid of other patients, which indicates that GSDME protein in synovial fluid can be used as a biomarker for the diagnosis of osteoarthritis, and the GSDME protein kit prepared by the invention conveniently and effectively realizes the detection of the expression level of GSDME protein in synovial fluid samples.

[0039] The present invention first discovered that the expression level of GSDME protein in osteoarthritis OA subtype is higher than that in samples without arthritis, proving that GSDME protein can be used as a biomarker for diagnosing osteoarthritis. Further, the clinical use value of the GSDME protein kit prepared by the present invention is verified, and the GSDME protein kit can be used to conveniently and quickly detect the expression level of GSDME protein in joint fluid samples.

[0040] Other experimental tests:

[0041] The present invention also conducted a cell experiment, taking chondrocytes from the joints of several subjects who did not suffer from arthritis, and using the enzyme-linked immunosorbent assay (ELISA) method to measure the expression level of the GSDME protein in the supernatant of the chondrocytes; then placing the chondrocytes under pyroptosis stimulation conditions, and using the enzyme-linked immunosorbent assay (ELISA) method to measure the expression level of the GSDME protein in the supernatant of the chondrocytes. The results showed that the expression level of the GSDME protein in the supernatant of the chondrocytes under the pyroptosis stimulation conditions was significantly higher than the expression level of the GSDME protein in the supernatant of the chondrocytes under normal conditions.

[0042] Experiments have shown that under pyroptosis stimulation, that is, in the course of osteoarthritis, there will be loss of cartilage matrix and death of chondrocytes, and a large amount of cell contents will be released into the synovial fluid, so the non-secretory protein GSDME protein highly expressed in the cytoplasm of the cell contents will be released into the synovial fluid; secondly, the osteoarthritis environment has oxidative stress conditions, which will act as an extracellular stimulus to induce GSDME protein-mediated cell pyroptosis, cell membrane lysis and destruction, and GSDME protein will be directly released into the blood in large quantities. The expression level of GSDME protein can be detected through synovial fluid.

[0043] The present invention also conducted biological analysis and clinical performance evaluation:

[0044] 1) Cartilage tissue and synovial fluid from the joints of nearly 100 patients with OA subtype and other patients in a hospital were collected, and the following biological analysis (transcriptome sequencing RNA-Seq technology, real-time fluorescence quantitative PCR method and enzyme-linked immunosorbent assay ELISA method) and clinical manifestation evaluation (clinical feature comparison) were performed; other patients were patients with other diseases besides arthritis, and cartilage tissue and synovial fluid from the joints were also required to be extracted for subsequent testing during the diagnosis process.

[0045] 2) Use RNA-Seq technology to process cartilage tissue from patients with OA subtypes of osteoarthritis to obtain transcriptome profiles, such as Figure 1 As shown in a), it is a single-center cartilage tissue differentially expressed gene transcriptome map of osteoarthritis OA subtypes, such as Figure 1 c) shows the transcriptome profile of differentially expressed genes in cartilage tissues of multiple centers of osteoarthritis OA subtypes;

[0046] 3) Cluster analysis was used to process the transcriptome maps of differentially expressed genes in single-center cartilage tissue and multi-center cartilage tissue, and the OA subtypes were divided into 1-4 categories (Cluster 1-4: glycosaminoglycan metabolism disorder subtype (Cluster 1), collagen metabolism disorder subtype (Cluster 2), activated sensory neuron subtype (Cluster 3) and inflammatory subtype (Cluster 4)). The OA subtypes in categories 1-4 correspond to the degree of clinical manifestation deterioration from low to high.

[0047] 4) Real-time fluorescence quantitative PCR was used to detect the cartilage tissue of patients with osteoarthritis OA subtype and other patients to obtain the expression level of GSDME mRNA in cartilage tissue, such as Figure 1 As shown in b), the single-center cartilage tissue GSDME mRNA expression levels of other patients (Normal) and osteoarthritis OA subtypes (Cluster 1-4) are shown. Figure 1 d) shows the expression level of GSDME mRNA in cartilage tissue of other patients (Normal) and osteoarthritis OA subtypes (Cluster 1-4), that is, the expression level of GSDME protein in cartilage tissue of patients with osteoarthritis OA subtypes;

[0048] 5) Use ELISA to measure the expression level of GSDME protein in the synovial fluid of patients with osteoarthritis (OA);

[0049] 6) Clinical manifestations were evaluated and clinical characteristics of patients with osteoarthritis OA subtypes in Cluster 1-3 and Cluster 4 were compared: WOMAC score and VAS score were used for clinical characteristics comparison ( Figure 1 f)), Kellgren-Lawrence (KL) grading system ( Figure 1 g)), osteophyte score ( Figure 1 h)), joint space score ( Figure 1 i)) and synovitis score ( Figure 1 j));

[0050] Through the above steps, the correlation between the expression level of GSDME protein in the cartilage tissue and synovial fluid of the joints of patients with osteoarthritis OA subtype and the degree of deterioration of clinical manifestations was analyzed. The results showed that GSDME protein was highly expressed in the cartilage tissue and synovial fluid of the joints of patients with Cluster 4 (OA inflammatory subtype). Figure 1 a)-e)) and is accompanied by a worsening of the clinical manifestations ( Figure 1 f)-j)). Figure 1 a)-e) of Cluster 4 (OA inflammatory subtype) showed that the expression level of GSDME protein was significantly higher than that of other patients, such as Figure 1 f)-j) show that patients with high GSDME protein expression levels in Cluster 4 (OA inflammatory subtype) have more severe clinical manifestations, proving that GSDME protein can be used as a biomarker for osteoarthritis.

[0051] Increased expression levels of GSDME protein in the synovial fluid of patients with the OA subtype of osteoarthritis are accompanied by worsening clinical manifestations. The expression level of GSDME protein in patients with the OA subtype of osteoarthritis is 2-5 times that of GSDME protein in other patients (i.e., patients without arthritis).

[0052] The present invention also conducted an animal experiment, dividing a number of healthy wild-type C57 mice into two groups, namely a medial meniscus instability DMM surgery group and a sham surgery group, and performing medial meniscus instability DMM surgery and sham surgery respectively, and establishing a C57 mouse osteoarthritis model; the medial meniscus instability DMM surgery group is referred to as the surgery group; the synovial fluid, cartilage tissue and knee joint of the surgery group and the sham surgery group in the C57 mouse osteoarthritis model were collected two months after the surgery:

[0053] a) extracting synovial fluid from the surgical group and the sham-operated group, and detecting the expression level of GSDME protein in the synovial fluid of the two groups using ELISA;

[0054] b) The knee joints of the surgical group and the sham-operated group were extracted and made into paraffin sections, and the expression levels of GSDME protein in the paraffin sections of the two groups were detected using immunofluorescence technology, such as Figure 2 As shown;

[0055] c) Cartilage tissues of the surgical group and the sham-operated group were extracted and ground, and the expression levels of GSDME protein in the ground cartilage tissues of the two groups were detected by Western blotting. Figure 3 As shown;

[0056] Through the above animal experiments, the results showed that wild-type C57 mice suffered from arthritis after undergoing DMM surgery for medial meniscus instability, and the expression levels of GSDME protein in their synovial fluid, cartilage tissue and knee joints were higher than those in the sham surgery group.

[0057] Among them, in the above b), after using the immunofluorescence technology, it was found that the fluorescence signal of GSDME protein was stronger in the mice in the surgery group, indicating that the expression level of GSDME protein in the knee joint of the surgery group was higher than that of the sham surgery group, and GSDME protein has the characteristic of high expression in the knee joint.

[0058] In the above c), after using the immunoblotting WB method for detection, the comparison protein detection bands of the surgical group and the sham operation group were obtained, such as Figure 3 In the GSDME protein, GSDME-FL is the full-length GSDME, and GSDME-N is the activated GSDME, both of which are correlated with the occurrence of osteoarthritis; among them, GAPDH is the internal reference protein to reflect the protein concentration. The depth of the contrast protein detection band was measured using imageJ software to characterize the relative expression level of GSDME protein in the surgical group and the sham operation group. The results showed that the expression level of GSDME-N (activated GSDME) in the cartilage tissue of the surgical group was higher than that in the sham operation group.

[0059] In the present invention, the application of GSDME protein as a biomarker in the diagnosis and prognosis evaluation of osteoarthritis is as follows:

[0060] 1. Diagnosis of osteoarthritis:

[0061] Construct a diagnostic kit for detecting osteoarthritis. Collect the patient's synovial fluid, use the kit to detect the expression level of GSDME protein in the synovial fluid, and feedback the detection results of GSDME protein. Finally, diagnose osteoarthritis based on the expression level of GSDME protein in the synovial fluid.

[0062] 2. Prognosis assessment of osteoarthritis:

[0063] (1) Establish a quantitative detection method for joint fluid GSDME protein: Use the constructed kit to detect the expression level of GSDME protein using enzyme-linked immunosorbent assay (ELISA).

[0064] (2) For patients who have been confirmed to have arthritis, joint fluid is collected and sent to the laboratory for testing.

[0065] (3) The laboratory uses the GSDME protein expression detection kit prepared by the invention to quantitatively detect the expression level of the GSDME protein in the synovial fluid, and feeds back the detection results of the GSDME protein.

[0066] (4) To make a prognostic assessment of the therapeutic effect of osteoarthritis based on the real-time expression level of GSDME protein in synovial fluid.

[0067] (5) During the treatment process, the real-time expression level of GSDME protein in the synovial fluid is monitored in real time to achieve real-time monitoring of the therapeutic efficacy of OA patients and timely adjust the drug type and its therapeutic dose to achieve the best therapeutic effect.

[0068] The present invention demonstrates at the cellular, animal and human levels that the expression level of GSDME increases significantly after osteoarthritis, verifying the feasibility of GSDME as a biomarker for detecting osteoarthritis; establishes a practical, sensitive and specific chemiluminescent immunological detection method for GSDME in synovial fluid; through clinical sample trials, verifies that GSDME in synovial fluid can be used to diagnose the severity of arthritis, and evaluates the clinical value of synovial fluid GSDME as a marker for evaluating the severity and diagnosis and prognosis of osteoarthritis; therefore, osteoarthritis can be diagnosed and predicted by detecting the expression level of GSDME in the synovial fluid of individual subjects, providing a new approach for the diagnosis of arthritis.

[0069] The present invention provides GSDME in joint fluid as a biomarker for detecting the severity and prognosis of osteoarthritis, and / or for use in a detection kit for evaluating the severity and efficacy of osteoarthritis.

[0070] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. Application of the biomarker GSDME, characterized by: The biomarker GSDME protein is used in preparing a kit for diagnosing osteoarthritis OA inflammatory subtypes, and the kit detects the biomarker GSDME protein in joint fluid.

2. The use of the biomarker GSDME according to claim 1, characterized in that: The biomarker GSDME protein is gasdermin E protein.

3. The use of the biomarker GSDME according to claim 1, characterized in that: The kit contains detection reagents and detection tools for detecting the expression level of the biomarker GSDME protein. The detection reagents include: human GSDME protein standard, anti-GSDME antibody, rabbit anti-human IgG H&G antibody, color reaction substrate solution and reaction termination solution; the detection tools include: ELISA plate.

4. The use of the biomarker GSDME according to claim 3, characterized in that: The human GSDME protein standard is specifically recombinant human GSDME protein; The color development reaction substrate solution is specifically a tetramethylbenzidine solution; The reaction termination solution is specifically a 2M sulfuric acid solution.

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