Osteoarthritis marker LOXL1 and its application

Abstract

The present invention belongs to the technical field of disease markers, and specifically relates to an osteoarthritis marker LOXL1 and its application. The osteoarthritis marker is the LOXL1 gene, and the amino acid sequence encoded by it is shown in SEQ ID NO.1. The present invention screens out a new OA inflammation marker LOXL1 through multi-data set collection screening. The AUC of the LOXL1 gene for predicting OA is 0.963, and the transcriptome RNA-seq data set verifies that LOXL1 is highly expressed in OA patients; LOXL1 performs ROC analysis on the validation group data set, and its AUC is 0.935; its gene and protein are highly expressed in OA patients, and LOXL1 has high diagnostic efficacy and clinical practical significance.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease markers, and in particular relates to an osteoarthritis marker and application thereof. Background Art

[0002] Osteoarthritis (OA) is the most common form of arthritis worldwide, affecting approximately 40% of people over the age of 70. The current diagnosis of OA is based on clinical symptoms of joint pain, stiffness, and loss of range of motion, as well as radiographic evidence of the disease, such as joint space narrowing, subchondral sclerosis, cyst formation, and osteophyte development, which usually appear in the late irreversible stage. Therefore, the "gold standard" for the clinical diagnosis of OA remains imaging. However, measurement of joint space width on plain radiographs usually requires serial assessments over 1-3 years; magnetic resonance imaging (MRI) is a more sensitive imaging method that can visualize cartilage, bone, synovial effusions, and ligaments, but its availability is limited due to the cost of MRI. Arthroscopy allows direct visualization of the cartilage surface, but even minimally invasive procedures are not routinely used in a large number of patients.

[0003] In recent years, the role of biomarkers in OA diagnosis, disease progression, and treatment has gained attention. Traditional laboratory tests, such as erythrocyte sedimentation rate and C-reactive protein (CRP) levels, provide some useful information about the general inflammatory process in OA patients, but these markers are not specific for joint inflammation and are poorly correlated with cartilage damage at the individual level.

[0004] At present, candidate biomarkers for OA are mainly focused on structural molecules (or their fragments) of bone, cartilage, and synovium, including type I collagen, type II collagen decomposition and metabolites, cartilage oligomeric matrix protein (COMP), matrix metalloproteinases (MMP), etc. Considering the limitations of the current available OA assessment, increasing evidence shows the key role of inflammatory biomarkers in the pathogenesis of osteoarthritis. Moreover, high-sensitivity C-reactive protein (hsCRP) has been shown to be elevated in the early stages of OA, indicating that inflammation is involved in the pathophysiological role of the early stages of the disease. If OA inflammatory biomarkers can be identified early and early intervention can be performed, the sequelae of cartilage and bone damage that lead to clinical disability can be delayed. Therefore, OA inflammatory biomarkers can be used as both a screening tool for OA and a cornerstone of treatment. Summary of the invention

[0005] In response to the above problems, the present invention screened out a new OA inflammation marker LOXL1 through screening of multiple data sets. The diagnostic efficacy (ROC) of LOXL1 gene for OA was 0.963, and LOXL1 was verified to be highly expressed in OA patients on the transcriptome RNA-seq data set; LOXL1 performed ROC analysis on the validation group data set, and its AUC was 0.935; its gene and protein were highly expressed in OA patients, and LOXL1 had high diagnostic efficacy and clinical significance.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides an osteoarthritis marker LOXL1, which is the LOXL1 gene, and the amino acid sequence encoded by the LOXL1 gene is shown in SEQ ID NO.1.

[0008] Another aspect of the present invention provides a specific substance that can specifically identify the osteoarthritis marker of the present invention.

[0009] In another aspect, the present invention provides a diagnostic reagent for diagnosing osteoarthritis, which comprises the specific substance of the present invention.

[0010] In another aspect, the present invention provides a diagnostic kit for diagnosing osteoarthritis, which comprises the specific substance of the present invention or the diagnostic reagent for diagnosing osteoarthritis of the present invention.

[0011] Another aspect of the present invention provides an inhibitor for inhibiting the osteoarthritis marker LOXL1 in the present invention.

[0012] In another aspect, the present invention provides a drug for treating osteoarthritis, which comprises the specific substance or the inhibitor of the present invention.

[0013] In another aspect, the present invention provides a use of the specific substance of the present invention in the preparation of a diagnostic product for diagnosing osteoarthritis.

[0014] In another aspect, the present invention provides use of the specific substance or the inhibitor of the present invention in the preparation of a medicament for treating osteoarthritis.

[0015] The beneficial effects of the present invention include at least:

[0016] (1) The present invention screened a new OA inflammatory marker LOXL1 through screening of multiple data sets. The diagnostic efficacy (AUC) of LOXL1 gene for OA was 0.963. The transcriptome RNA-seq data set verified that LOXL1 was highly expressed in OA patients. ROC analysis of LOXL1 on the validation group data set showed an AUC of 0.935. Its gene and protein were highly expressed in OA patients. LOXL1 has high diagnostic efficacy and clinical significance.

[0017] (2) The present invention has confirmed through a series of experiments that the expression of LOXL1 in OA patients is positively correlated with cytokines IL-6 and IL-8; the expression of LOXL1 and IL-6 in the cartilage of osteoarthritis rats is increased, and synovial iNOS + The expression of M1 macrophages increased, and LOXL1 was positively correlated with the expression of IL-6 and iNOS. In vitro, LOXL1 stimulated macrophages to highly express CD86 and increase the secretion of IL-1β, IL-6, and TNF-α. LOXL1 was closely related to the expression of inflammatory cytokines and the activation of inflammatory macrophages in OA patients. Chondrocytes can communicate with macrophages through LOXL1 and jointly participate in the occurrence and progression of OA inflammatory response. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The differentially expressed genes between osteoarthritis cartilage tissue and the control group, where red indicates up-regulation and blue indicates down-regulation;

[0019] Figure 2 Nine characteristic genes were obtained for LASSO algorithm screening;

[0020] Figure 3 Twenty-five signature genes were obtained for SVM-RFE screening;

[0021] Figure 4 It is the Venn diagram of 8 overlapping genes screened by LASSO regression and SVM-RFE algorithm;

[0022] Figure 5 is the ROC curve of LOXL1 gene in the training group;

[0023] Figure 6 To verify the expression of LOXL1 gene on RNA-seq dataset;

[0024] Figure 7 is the ROC curve of LOXL1 in the RNA-seq dataset;

[0025] Figure 8 Comparison of 22 immune cell subtypes between osteoarthritis and control groups, where blue and red represent normal and osteoarthritis samples, respectively;

[0026] Fig. 9 The correlation between LOXL1 and infiltrating immune cells in osteoarthritis tissues;

[0027] Fig.10 is the expression of LOXL1 mRNA in whole blood of OA patients;

[0028] Fig.11 is the expression of LOXL1 protein in the serum of OA patients;

[0029] Fig.12 Immunohistochemical analysis of LOXL1, IL-6 and IL-10 in the knee cartilage tissue of OA rats and the control group (CTR) (scale bar = 100 μm);

[0030] Fig.13 Quantitative analysis of LOXL1, IL-6 and IL-10 in the knee cartilage tissue of OA rats and the control group (CTR), among which, *P<0.05, **P<0.01, ***P<0.001;

[0031] Fig.14 Immunofluorescence analysis of iNOS, Arg1 and CD11b in the synovium of the knee joints of OA rats and the control group (CTR) (scale bar = 25 μm);

[0032] Fig.15 Quantitative analysis of iNOS, Arg1 and CD11b in the synovium of the knee joints of OA rats and the control group (CTR), among which, *P<0.05, **P<0.01, ***P<0.001;

[0033] Fig.16 The morphology of macrophages in the control group (CTR) and the LOXL1 intervention group under the microscope;

[0034] Fig.17 Flow cytometry was used to detect the expression of macrophage CD86 and CD206 in the control group and LOXL1 intervention group;

[0035] Fig.18 The expression of various inflammatory factors in the cell culture supernatant of the control group and the LOXL1 intervention group, among which *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0036] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0037] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, materials, or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, materials, or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0038] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0039] The embodiment of the present invention provides an osteoarthritis marker LOXL1, which is the LOXL1 gene, and the amino acid sequence encoded by the LOXL1 gene is shown in SEQ ID NO.1.

[0040] Specifically, the sequence of SEQ ID NO.1 is as follows:

[0041] MALARGSRQLGALVWGACLCVLVHGQQAQPGQGSDPARWRQLIQWENNGQVYSLL

[0042] NSGSEYVPAGPQRSESSSRVLLAGAPQAQQRRSHGSPRRRQAPSLPLPGRVGSDTVRGQAR

[0043] HPFGFGQVPDNWREVAVGDSTGMARARTSVSQQRHGGSASSVSASAFASTYRQQPSYPQ

[0044] QFPYPQAPFVSQYENYDPASRTYDQGFVYYRPAGGGVGAGAAAVASAGVIYPYQPRARY

[0045] EEYGGGEELPEYPPQGFYPAPERPYVPPPPPPPDGLDRRYSHSLYSEGTPGFEQAYPDPGPE

[0046] AAQAHGGDPRLGWYPPYANPPPEAYGPPRALEPPYLPVRSSDTPPPGGERNGAQQGRLSV

[0047] GSVYRPNQNGRGLPDLVPDPNYVQASTYVQRAHLYSLRCAAEEKCLASTAYAPEATDYD

[0048] VRVLLRFPQRVKNQGTADFLPNRPRHTWEWHSCHQHYHSMDEFSHYDLLDAATGKKVA

[0049] EGHKASFCLEDSTCDFGNLKRYACTSHTQGLSPGCYDTYNADIDCQWIDITDVQPGNYILK

[0050] VHVNPKYIVLESDFTNNVVRCNIHYTGRYVSATNCKIVQS.

[0051] It should be noted that the current research on OA markers is mainly focused on the structural components of the cartilage matrix, especially the degradation markers of type II collagen. Although some biological markers of joint metabolism may be significantly increased on a population basis in patients with early OA, these markers cannot be used as a diagnostic test for OA in individual patients and cannot reflect the inflammatory status of OA patients. In addition, in addition to the identification of new inflammatory biomarkers, it is also very important to study the role of specific biomarkers in the pathogenesis of OA. The most abundant immune cells in the joints are macrophages, which are considered to be key players in the onset and progression of OA. Macrophages in the synovium can degrade the extracellular matrix of cartilage cells by releasing inflammatory molecules, promoting the onset of OA and its inflammatory response. Removal of CD 14 in synovial cell culture + Macrophages can reduce the expression of IL-1β, TNF-α, MMPs, and aggrecanase that degrade articular cartilage. Macrophage-related inflammation is a driver of OA structural damage and progression. Soluble markers of activated macrophages are closely associated with the severity of knee inflammation and the risk of OA progression. By detecting markers of macrophage inflammatory pathways, radiation exposure imaging tests for OA patients can be avoided, which has clinical practical prospects; moreover, targeting macrophages and macrophage-related inflammatory pathways may be a way to reduce OA symptoms and joint structural deterioration, which is of great significance for the diagnosis and treatment of OA.

[0052] The present invention is based on the GEO database in R language, and screens out the characteristic gene LOXL1 expressed by chondrocytes and closely related to osteoarthritis through bioinformatics and machine learning. Lysyl oxidase-like 1 (LOXL1), a protein encoded by the LOXL1 gene, is a protein belonging to the collagen fiber cross-linking enzyme family, which plays an important role in maintaining tissues rich in extracellular matrix (ECM). The present invention found that the AUC of the LOXL1 gene for predicting OA is 0.963 (95% CI is 0.867-1.000). The transcriptome RNA-seq data set verifies that LOXL1 is highly expressed in OA patients; LOXL1 performs ROC analysis on the validation group data set, and its AUC is 0.935 (95% CI is 0.843-0.994); there are significant differences in the infiltration of NK cells and macrophages between the OA and control groups, and macrophages are significantly correlated with LOXL1, and LOXL1 may play a potential role in the occurrence and development of OA. The serum of OA and normal healthy control groups was collected. The expression of LOXL1 gene and protein in OA patients was higher than that in normal healthy control group, and was positively correlated with cytokines IL-6 and IL-8 in the serum of OA patients. Compared with normal rats, the expression of LOXL1 and IL-6 in the cartilage of osteoarthritis rats was increased, and the synovial iNOS + The expression of LOXL1 in M1 macrophages was increased, and LOXL1 was positively correlated with the expression of IL-6 and iNOS. In vitro, LOXL1 stimulated macrophages to highly express CD86 and increase the secretion of IL-1β, IL-6, and TNF-α.

[0053] Another embodiment of the present invention provides a specific substance that can specifically identify the osteoarthritis marker of the present invention. It should be noted that the specific substance that specifically identifies the osteoarthritis marker of the present invention includes a binding polypeptide that can bind to the protein or a substance that can develop color with the protein.

[0054] Another embodiment of the present invention provides a diagnostic reagent for diagnosing osteoarthritis, which includes the specific substance of the present invention. It should be noted that the above-mentioned specific substance can be prepared into a diagnostic reagent for diagnosing osteoarthritis for the diagnosis of osteoarthritis.

[0055] Another embodiment of the present invention provides a diagnostic kit for diagnosing osteoarthritis, which includes the specific substance of the present invention or the diagnostic reagent for diagnosing osteoarthritis of the present invention. It should be noted that in addition to preparing the specific substance into a diagnostic reagent, it can also be prepared into a diagnostic kit.

[0056] Another embodiment of the present invention provides an inhibitor for inhibiting the osteoarthritis markers of the present invention.

[0057] Still another embodiment of the present invention provides a drug for treating osteoarthritis, which comprises the specific substance of the present invention or the inhibitor of the present invention.

[0058] It should be noted that the inhibitor used to inhibit the osteoarthritis marker of the present invention or the specific substance of the present invention that specifically recognizes the osteoarthritis marker of the present invention can achieve the purpose of treating osteoarthritis by inhibiting the expression of the osteoarthritis marker or competitively binding to the marker.

[0059] Still another embodiment of the present invention provides a use of the specific substance of the present invention in preparing a diagnostic product for diagnosing osteoarthritis.

[0060] In some specific embodiments, the diagnostic product in the above application includes a diagnostic reagent or a diagnostic kit.

[0061] Still another embodiment of the present invention provides a use of the specific substance or the inhibitor of the present invention in the preparation of a drug for treating osteoarthritis.

[0062] In some specific embodiments, the dosage form of the drug in the above application includes tablets, powders, capsules, pills, injections, oral liquids or emulsions.

[0063] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0064] Example 1 Differential gene screening and functional verification

[0065] (1) Screening of differentially expressed genes

[0066] The GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ) was used to screen out data sets containing human osteoarthritis cartilage samples and control group cartilage samples, and three chip data sets were obtained: GSE178557, GSE183531, and GSE169077, and one transcriptome RNA-seq data set: GSE114007; among them, GSE178557, GSE183531, and GSE169077 were used as training groups for data merging to obtain 15 osteoarthritis cartilage samples and 9 control group cartilage samples. At the same time, GSE114007 was used as an independent validation group to obtain 20 osteoarthritis cartilage samples and 18 control group cartilage samples. The SVA package of R language was used for preprocessing and batch effect removal, and the limma package was used for differential analysis; P < 0.05 and absolute value of difference fold > 2 were used as screening criteria for differentially expressed genes. Finally, 32 differentially expressed genes were screened, including 9 up-regulated genes and 23 down-regulated genes (see Figure 1).

[0067] (2) Screening of candidate diagnostic markers

[0068] In order to further obtain characteristic markers related to osteoarthritis, the present invention uses two machine learning algorithms, LASSO regression and SVM-RFE, to further screen the gene set (see Figure 2 , Figure 3 ), and took the overlapping genes of the two, and finally obtained 8 genes, including LOXL1, HILPDA, IRAK3, BEST1, HIST1H1C, CYP3A5, FAM65B and HSD11B1 ( Figure 4 ). It was tested on the independent validation set GSE114007, and the change trends of 5 genes were consistent with the training set, including LOXL1, HSD11B1, BEST1, HILPDA and HIST1H1.

[0069] (3) The diagnostic value of biomarkers for osteoarthritis

[0070] To test the predictive value of the above biomarkers, the ROC curve was generated using the mRNA expression level of the training group samples, and the ROC curve, i.e., the AUC value, was used to determine the diagnostic value of the gene in identifying osteoarthritis. The same method was used to test the validation group GSE114007. The results are shown in Figure 2. Figure 5 , Figure 6 and Figure 7 As shown, the results showed that LOXL1 had a strong identification ability (AUC>0.9) in the above datasets.

[0071] (4) Study on infiltration of various subtypes of immune cells

[0072] The present invention uses the bioinformatics algorithm CIBERSORT (https: / / cibersortx.stanford.edu / ) to quantify the relative proportion of infiltrating immune cells in the osteoarthritis gene expression profile, and estimates the abundance of immune cells using a reference set of 22 immune cell subtypes (LM22) and 1000 permutations. The results are shown in Figure 8 and Fig. 9 As shown, the results showed that there were significant differences in macrophages between osteoarthritis cartilage tissue and normal control cartilage tissue, and the number of macrophages in osteoarthritis cartilage was significantly higher than that in the control group, P<0.001.

[0073] Example 2 Expression of LOXL1 in the blood of OA patients

[0074] All participants in the trial were informed of the research process and sample use and signed informed consent. The demographic and clinical data of all participants were obtained through questionnaire collection, physical examination and blood sampling. There was no statistically significant difference in the general information of the patients.

[0075] (1) Detection of LOXL1 mRNA expression in whole blood

[0076] Peripheral blood of 15 subjects was collected for the detection of LOXL1 mRNA expression in whole blood, including samples from 7 osteoarthritis patients and 8 normal control samples. RNA was extracted using a blood RNA extraction kit (beyotime, China), reverse transcription was performed according to the instructions of HiScript IIQ RT SuperMix for qPCR (vazyme, China), and real-time fluorescence quantitative PCR of LOXL1 was performed using ChamQ Universal SYBR qPCR Master Mix (vazyme, China). The relative quantification method was used to compare the differences in LOXL1 expression. The results are shown in Figure 2. Fig.10 As shown, the results showed that the mRNA expression level of LOXL1 in the whole blood of osteoarthritis patients was significantly higher than that of the control group.

[0077] (2) Detection of LOXL1 protein expression in serum

[0078] Serum from 24 subjects was collected, including 12 samples from osteoarthritis patients and 12 samples from the normal control group, and stored at -80°C for later use. The ELISA kit instructions (CUSABIO, China) were followed to read the optical density at 450nm, draw a concentration standard curve, and calculate the LOXL1 concentration. Fig.11 As shown, the results showed that the protein expression of LOXL1 in the serum of osteoarthritis patients was significantly higher than that in the control group.

[0079] Example 3 Expression of inflammatory factors in serum of OA patients

[0080] The above-mentioned frozen serum of the subjects was used to detect the expression of each inflammatory factor (IL-6, IL-8, IL-10) using a cytokine detection kit (BIOPREDIA, China), and the correlation analysis between LOXL1 and cytokine expression levels was performed. The results are shown in Table 1 below, and the results show that LOXL1 is positively correlated with IL-6 and IL-8, and is statistically significant, and the correlation with IL-10 is not statistically significant.

[0081] Table 1 Correlation between serum LOXL1 and protein expression of various inflammatory factors

[0082] Inflammatory factors LOXL1 IL-4 IL-6 IL-8 IL-10 LOXL1 1 - - - - IL-4 0.286 1 - - - IL-6 0.407* 0.394 1 - - IL-8 0.682** 0.26 0.296 1 IL-10 0.164 0.660** 0.597** 0.063 1

[0083] *P<0.05, **P<0.01

[0084] Example 4 Expression of LOXL1, synovial macrophages and inflammatory factors in cartilage of osteoarthritis rats

[0085] (1) Establishment of osteoarthritis rat model

[0086] Four-week-old female SD rats were randomly divided into an experimental group and a control group, and anterior cruciate ligament resection (ACLT) was used to construct an animal model of SD rat osteoarthritis. This model simulates the pathological changes of human knee osteoarthritis by surgically removing the anterior cruciate ligament of the knee joint of the rats in the experimental group. During the operation, appropriate anesthetic drugs were used to ensure that the rats were in a painless and unconscious state. Local disinfection and aseptic operation were performed in the knee joint area of ​​the rats. The patellar ligament was exposed through an incision. After cutting the synovial tissue of the knee joint, the anterior cruciate ligament of the rat was cut with a scalpel without damaging the surrounding joint structure. Finally, the surgical incision was properly sutured to ensure good wound healing.

[0087] (2) Detection of LOXL1 expression in rat cartilage

[0088] After 9 weeks of constructing the osteoarthritis model of SD rats using ACLT, the rats were killed with 1% sodium pentobarbital (50 mg / kg), and the cartilage tissues of the experimental and control groups were collected. The cartilage tissues were fixed in 4% paraformaldehyde for 24 hours; 10% EDTA decalcification solution was used for constant temperature shaker decalcification, and the decalcification solution was replaced every 2-3 days. The probe punctured the bone tissue without resistance, and the penetration of the tissue was the standard for decalcification completion; after decalcification, it was rinsed under running water overnight, and gradient alcohol dehydration was performed in a dehydrator, 75% alcohol for 2 hours, 85% alcohol for 2 hours, 90% alcohol for 1.5 hours, 95% alcohol for 2 hours, anhydrous ethanol I for 2 hours, anhydrous ethanol II for 2 hours, alcohol benzene for 40 minutes, xylene I for 40 minutes, xylene II for 40 minutes, 6 Melt paraffin I at 5° for 0.5h, melt paraffin II at 65° for 1h, and melt paraffin III at 65° for 2h45min; after dehydration and wax immersion, embed the wax-immersed tissue in an embedding machine, and then place the wax block on a paraffin slicer to slice with a thickness of 4μm. Use a 40℃ slide spreader to pick up the slices and bake them at 60℃; then perform antigen repair, and after natural cooling, place the slides in PBS (PH7.4) and shake on a decolorizing shaker to wash 3 times, each time for 5min; place the slices in a 3% hydrogen peroxide solution and incubate them at room temperature in the dark for 25min to block endogenous peroxidase, and place the slides on P Wash 3 times with PBS (PH7.4) on a decolorizing shaker, 5 min each time; cover the tissue evenly with 3% BSA and block at room temperature for 30 min; discard the blocking solution, add an appropriate amount of LOXL1 primary antibody, and incubate in a humidified box at 4°C overnight; wash the slides 3 times with PBS (PH7.4) on a decolorizing shaker, 5 min each time, then add a secondary antibody (HRP labeled) of the same species as the primary antibody to cover the tissue and incubate at room temperature for 50 min; wash the slides 3 times with PBS (PH7.4) on a decolorizing shaker, 5 min each time, and slice them slightly. After drying, add freshly prepared DAB color developing solution in the circle, control the color development time under a microscope, the positive color is brown-yellow, and rinse the slices with tap water to stop the color development; counterstain with hematoxylin for about 3 minutes, wash with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, turn blue with hematoxylin blueing solution, and rinse with running water; put the slices in 75% alcohol for 5 minutes, 85% alcohol for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, n-butanol for 5 minutes, and xylene I for 5 minutes to dehydrate and make them transparent, take the slices out of xylene and dry them slightly, and seal them with sealing glue; examine under a microscope.

[0089] The results are as follows Fig.12 As shown, a bar graph is drawn as Fig.13 As shown, the results showed that the expression level of LOXL1 in the cartilage tissue of osteoarthritis rats was significantly higher than that in the control group.

[0090] (3) Detection of the expression levels of iNOS and Arg1 in synovial macrophages M1 and M2

[0091] Nine weeks after the osteoarthritis model of SD rats was established by ACLT, the mice were killed with 1% sodium pentobarbital (50 mg / kg), and the synovial tissues of the rats in the experimental group and the control group were collected. The synovial tissue was fixed in 4% paraformaldehyde for 24 hours; dehydrated in a dehydrator with gradient alcohols, 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, alcohol benzene for 10 minutes, xylene I for 10 minutes, xylene II for 10 minutes, 65° melted paraffin I for 1 hour, 65° melted paraffin II for 1 hour, 65° melted paraffin III for 1 hour; the wax-soaked tissue was embedded in an embedding machine, and then the wax block was placed in a paraffin slicer for slicing, the slice thickness was 4μm, the slice was picked up by a 40℃ slide spreader, and the slice was dried at 60℃; the slices were placed in environmentally friendly dewaxing liquid for 10 minutes, environmentally friendly dewaxing liquid for 10 minutes, environmentally friendly dewaxing liquid for 10 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, anhydrous ethanol III for 5 minutes, and washed with distilled water; antigen repair was then performed, and after natural cooling, the slides were placed in PBS (PH7.4 ) and washed 3 times on a decolorizing shaker with shaking, each time for 5 minutes; after the slices were slightly dried, a circle was drawn around the tissue with a tissue pen, and BSA was added in the circle for incubation for 30 minutes; the blocking solution was discarded, and two primary antibodies (iNOS and CD11b, Arg1 and CD11b) were mixed at a certain dilution ratio and dropped on the tissue, and the slices were placed flat in a wet box and incubated overnight at 4°C; the glass slides were placed in PBS (PH7.4) and washed 3 times on a decolorizing shaker with shaking, each time for 5 minutes, and after the slices were slightly dried, the secondary antibody labeled with the corresponding species of the primary antibody and prepared in a certain ratio was added in the circle to cover the tissue, and incubated at room temperature for 50 minutes; DAPI staining solution was added in the circle, and incubated at room temperature for 10 minutes in the dark; an autofluorescence quencher was added in the circle for 5 minutes, and washed with running water for 10 minutes; the glass slides were placed in PBS (PH7.4) and washed 3 times on a decolorizing shaker with shaking, each time for 5 minutes, and the slices were slightly dried and sealed with anti-fluorescence quenching sealing agent; microscopic examination.

[0092] The results are as follows Fig.14 As shown, a bar graph is made as Fig.15 As shown, the results showed that the expression level of iNOS, a protein representing macrophage polarization toward M1, in the synovial tissue of osteoarthritis rats was significantly higher than that in the control group, and there was no statistically significant change in the expression level of Arg1, a protein representing macrophage polarization toward M2.

[0093] (4) Detection of inflammatory factor expression in synovial tissue

[0094] After 9 weeks of constructing SD rat osteoarthritis model with ACLT, mice were killed with 1% sodium pentobarbital (50 mg / kg), and synovial tissues of rats in the experimental and control groups were collected. The synovial tissues were fixed in 4% paraformaldehyde for 24 hours; dehydrated in a dehydrator with gradient alcohols, 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, alcohol benzene for 10 minutes, xylene I for 10 minutes, xylene II for 10 minutes, 65° melted paraffin I for 1 hour, 65° melted paraffin II for 1 hour, 65° melted paraffin III for 1 hour; the wax-soaked tissues were embedded in an embedding machine, and then the wax blocks were placed in a paraffin slicer for sectioning, with a section thickness of 4 μm, 4 The slides were picked up in a slide spreader at 0℃ and dried at 60℃; the sections were placed in environmentally friendly dewaxing solution for 10 minutes, environmentally friendly dewaxing solution for 10 minutes, environmentally friendly dewaxing solution for 10 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, anhydrous ethanol III for 5 minutes, and washed with distilled water; antigen repair was then performed, and after natural cooling, the slides were placed in PBS (PH7.4) and washed on a decolorizing shaker for 3 times, each time for 5 minutes; the sections were placed in a 3% hydrogen peroxide solution and incubated at room temperature in the dark for 25 minutes to block endogenous peroxidase, and the slides were placed in PBS (PH7.4) Wash the slides 3 times on a decolorizing shaker with 5% BSA, each time for 5 minutes; evenly cover the tissue with 3% BSA and block at room temperature for 30 minutes; discard the blocking solution, add the corresponding primary antibodies (IL-6, IL-10) on the sections, and incubate them in a humidified box at 4°C overnight; wash the slides 3 times on a decolorizing shaker with PBS (PH7.4), each time for 5 minutes, then add the secondary antibody (HRP labeled) of the corresponding species to the primary antibody to cover the tissue, and incubate at room temperature for 50 minutes; wash the slides 3 times on a decolorizing shaker with PBS (PH7.4), each time for 5 minutes, and then cut the slides. After the slices were slightly dried, freshly prepared DAB coloring solution was added to the circle. The coloring time was controlled under a microscope. The positive color was brown-yellow. The slices were rinsed with tap water to stop the coloring. The slices were counterstained with hematoxylin for about 3 minutes, washed with tap water, differentiated with hematoxylin differentiation solution for a few seconds, rinsed with tap water, and blued with hematoxylin blueing solution. The slices were placed in 75% alcohol for 5 minutes, 85% alcohol for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, n-butanol for 5 minutes, and xylene I for 5 minutes to dehydrate and make them transparent. The slices were taken out of xylene and dried slightly, and sealed with sealing glue. The results showed that the expression of pro-inflammatory factor IL-6 in the synovial tissue of rats with osteoarthritis was significantly higher than that of the control group, and the expression of anti-inflammatory factor IL-10 was significantly lower than that of the control group.At the same time, the correlation analysis of LOXL1 expression level with iNOS, Arg1, IL-6 and IL-10 was performed. The results are shown in Table 2 below (where *P<0.05 indicates a significant difference). The results showed that LOXL1 was positively correlated with IL-6 and iNOS, and was statistically significant, while the correlation with Arg1 and IL-10 was not statistically significant.

[0095] Table 2 Correlation analysis between LOXL1 expression and iNOS, Arg1, IL-6 and IL-10

[0096] Inflammatory factors IL-6 IL-10 iNOS Arg1 LOXL1 0.587* 0.308 0.664* 0.196

[0097] Example 5 LOXL1 can stimulate macrophage polarization toward M1 phenotype and increase the expression of inflammatory cytokines

[0098] (1) THP-1 cells are induced into macrophages

[0099] In a sterile 6-well plate, 100 × 10 THP-1 cells were seeded. 5 100 ng / ml PMA solution was selected to induce THP-1 to differentiate into macrophages, and the cells were cultured in a 37°C, 50 mL / L CO2 saturated humidity incubator for 24 h. Cell adhesion was observed under a microscope as a sign that THP-1 was successfully induced into macrophages (see Fig.16 ).

[0100] (2) Cell grouping

[0101] The macrophages were divided into a control group and a LOXL1 intervention group, with 3 replicate wells in each group.

[0102] (3) LOXL1 stimulates macrophage polarization toward M1

[0103] Treat macrophages with exogenous LOXL1 protein at a concentration of 4 μg / ml, collect cells after 48 hours; resuspend with PBS and centrifuge, resuspend with Staining Buffer, add CD86 direct-labeled antibody (eBioscience, USA), and stain for 30 minutes in the dark; resuspend cells with Staining Buffer, centrifuge, repeat the operation once; resuspend cells completely with Staining Buffer; select appropriate channels for on-machine detection according to the fluorescent dye on the direct-labeled antibody, and use FlowJo software to analyze the cell positive rate and the average fluorescence intensity of the positive area. The results are shown in Fig.17 As shown, the results showed that the CD86 positivity rate of macrophages in the LOXL1 intervention group was significantly higher than that in the control group, indicating that LOXL1 stimulated macrophages to polarize toward M1.

[0104] (4) LOXL1 stimulates macrophages to increase the expression of pro-inflammatory factors

[0105] Macrophages were treated with exogenous LOXL1 protein at a concentration of 4 μg / ml, and the cell supernatant was collected 48 hours later. The expression levels of various inflammatory factors (IL-1β, IL-6, IL-10, TNF-α) were detected using a cytokine detection kit (BIOPREDIA, China). Fig.18 As shown, the results showed that the IL-1β, IL-6, and TNF-α secreted by macrophages in the LOXL1 intervention group were significantly higher than those in the control group, and there was no statistically significant change in the expression of IL-10.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. Use of a reagent for detecting an osteoarthritis marker LOXL1 in the preparation of a diagnostic product for osteoarthritis, wherein the osteoarthritis marker LOXL1 is the LOXL1 gene, and the amino acid sequence encoded by the LOXL1 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: Diagnostic products include diagnostic reagents or diagnostic kits.

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