Use of peptidyl prolyl cis / trans isomerases as markers for osteoarthritis

Abstract

The present application relates to a kind of biomarkers related to osteoarthritis, which is Pin1 protein, by detecting whether the expression level of Pin1 protein is abnormal, osteoarthritis can be diagnosed or osteoarthritis prognosis can be predicted, by testing the influence of drug on the expression level of Pin1 protein, osteoarthritis treating drug can be screened or prepared, the quality and efficacy of osteoarthritis treating or relieving drug can be evaluated.The present application uses TMT proteomics technology to complete the screening of biomarkers related to osteoarthritis, and finds the common intersection of 6 proteins in the results of osteoarthritis disease target, TMT proteomics and protein targeting verification through way analysis, wherein Pin1 is consistent with the trend of TMT proteomics and protein targeting verification, i.e.Pin1 is decreased in osteoarthritis.New biomarker related to osteoarthritis-Pin1 protein is found, which has important biological significance and pharmaceutical significance.

Description

Technical Field

[0001] This invention relates to the fields of diagnosis, treatment, and prognosis prediction. More specifically, it relates to the diagnosis of osteoarthritis or the prediction of osteoarthritis prognosis by detecting abnormalities in peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1). Furthermore, testing the effect of drugs on Pin1 protein expression levels can be used to screen or prepare drugs for treating osteoarthritis and to evaluate the quality and efficacy of drugs for treating or alleviating osteoarthritis. Background Technology

[0002] Osteoarthritis is a chronic, multifactorial joint disease characterized by progressive degeneration of articular cartilage.

[0003] Articular cartilage is a layer of smooth, elastic connective tissue covering the joint surface. It is highly elastic and wear-resistant, playing a vital role in transmitting loads, absorbing shocks, and lubricating the joint, thus maintaining its structural and functional stability. Articular cartilage is neither innervated by nerves nor contains blood vessels supplying oxygen or nutrients. The only cell type present in this tissue is chondrocyte, which plays a crucial role in cartilage formation, metabolism, and repair.

[0004] Because articular cartilage is an avascular structure, it does not cause clinical symptoms in the early stages until nerve-innervated tissues are involved. This is one of the reasons for the delayed diagnosis of osteoarthritis. By this time, the cartilage damage has already affected the subchondral bone. Therefore, developing biomarkers for the early diagnosis of osteoarthritis and applying them to clinical diagnosis is an urgent problem to be solved.

[0005] Rongjin Niantong Formula originates from a high-frequency, core herbal formula for treating osteoarthritis (bone pain) in the "Collection of Imperial Prescriptions" by Academician Chen Keji, a master of traditional Chinese medicine. It comprises six herbs: Achyranthes bidentata, Angelica sinensis, Angelica pubescens, Notopterygium incisum, Saposhnikovia divaricata, and Glycyrrhiza uralensis. Achyranthes bidentata is the principal herb, used to tonify the liver and kidneys and strengthen tendons and bones, while also promoting blood circulation to unblock the meridians of the limbs. Angelica sinensis is the assistant herb, nourishing and harmonizing the blood to invigorate tendons, bones, and blood vessels. Angelica pubescens effectively treats latent wind and eliminates chronic pain; Notopterygium incisum dispels wind and dampness, benefits joints, and relieves pain; Saposhnikovia divaricata dispels wind, eliminates dampness, and relieves pain. These three herbs, combined, eliminate pain throughout the body, serving as adjuvant herbs to dispel wind and dampness and relieve pain. Glycyrrhiza uralensis is effective in treating limb contractures, relieving spasms and pain, and also harmonizes the other herbs. Together, these herbs tonify the liver and kidneys, strengthen tendons and bones, dispel wind and dampness, and relieve pain, effectively treating the pathogenesis of knee osteoarthritis. Studies have shown that Rongjin Niantong Formula can effectively alleviate the clinical symptoms and signs of patients with knee osteoarthritis. A patent application filed on October 26, 2018, concerning the preparation process of Rongjin Niantong Formula (patent number 201810899834.9), also mentions that Rongjin Niantong Formula has good clinical efficacy in treating knee osteoarthritis, and that its active ingredients have a multi-target synergistic therapeutic effect on knee osteoarthritis. Therefore, it is evident that Rongjin Niantong Formula has many targets of action. It is necessary to provide more information on the targets of Rongjin Niantong Formula to make the pharmacological research of Rongjin Niantong Formula more comprehensive and richer, laying a foundation for further research on the specific mechanism of Rongjin Niantong Formula in treating osteoarthritis and the identification of biomarkers. Summary of the Invention

[0006] The purpose of this invention is to provide a biomarker related to osteoarthritis, namely Pin1 protein. By detecting whether there is an abnormality in the expression level of Pin1 protein, osteoarthritis can be diagnosed or its prognosis can be predicted. By testing the effect of drugs on the expression level of Pin1 protein, drugs for treating osteoarthritis can be screened or prepared, and the quality and efficacy of drugs for treating or alleviating osteoarthritis can be evaluated.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A biomarker associated with osteoarthritis, the biomarker being the Pin1 protein.

[0009] The use of a biomarker in the preparation of products for diagnosing osteoarthritis and / or assessing the risk of developing osteoarthritis, said biomarker being the Pin1 protein.

[0010] The products include chips or reagent kits.

[0011] The use of a biomarker in screening or preparing drugs for treating osteoarthritis, wherein the biomarker is Pin1 protein.

[0012] The application of the biomarkers in the preparation of reagents for evaluating the quality of drugs for the treatment or relief of osteoarthritis.

[0013] A biomarker related to the efficacy of Rongjin Niantong Formula in treating osteoarthritis (i.e., the target of Rongjin Niantong Formula in treating osteoarthritis), wherein the biomarker is Pin1 protein.

[0014] A method for screening biomarkers associated with osteoarthritis, comprising the following steps: (1) sample mixing and biological replication, (2) extraction and quantification of tissue proteins, (3) FASP digestion, (4) TMT labeling, (5) RP grading of peptides, (6) separation of peptides, and (7) identification and relative quantitative analysis of proteins.

[0015] The specific method for separating the polypeptide in step (6) is as follows: the polypeptide is separated after proteolytic digestion using nano-liquid chromatography-high resolution mass spectrometry; the chromatographic column is equilibrated with 0.1% formic acid solution for 30 min, and then eluted according to the separation conditions in the table below:

[0016]

[0017] The mobile phase A is 0.1% formic acid; the mobile phase B is a 0.1% formic acid-acetonitrile aqueous solution, with acetonitrile accounting for 84% by volume.

[0018] The high-resolution Q-Exactive mass spectrometer was used in positive ion mode with a scan range of 300-1800 m / z. Ten secondary fragment spectra were acquired after each full scan. The MS resolution was set to 70,000, and the MS / MS resolution was set to 17,500. The collision energy was 30 eV.

[0019] In step (7) protein identification and relative quantification, mass spectrometry data were identified and relative quantified using Masscot 2.2 and ProteomeDiscoverer 1.4.

[0020] The specific method for RP fractionation of the peptide in step (5) is as follows:

[0021] After labeling, the peptides were lyophilized and mixed in equal volumes. 100 μg of the peptide mixture was diluted with 300 μL of 0.1% TFA, separated by reversed-phase high-pH chromatography, and each eluent was collected. Each fraction was lyophilized and reconstituted with 12 μL of 0.1% formic acid. The absorbance of each peptide fraction was measured at 280 nm, and the concentration of each fraction was calculated.

[0022] The reversed-phase high-pH chromatographic conditions were: Thermo Fisher Easy nLC, with a Thermoscientific EASY column, 10cm, ID 75μm, 3μm, C18-A2.

[0023] In addition, the present invention also provides the application of reagents for detecting Pin1 protein in the preparation of tools for diagnosing osteoarthritis or predicting the prognosis of osteoarthritis.

[0024] The reagents for detecting Pin1 protein include those for detecting the expression level of Pin1 protein.

[0025] The reagent is a substance capable of binding to the Pin1 protein; the substance is capable of detecting the expression level of the Pin1 protein.

[0026] Compared with the prior art, the advantages of the present invention are as follows:

[0027] This invention utilizes TMT proteomics technology to screen biomarkers associated with osteoarthritis. Through Wayne analysis of osteoarthritis disease targets, TMT proteomics, and protein targeting validation results, six proteins with common intersections were identified. Among these, Pin1 showed a consistent trend in both TMT proteomics and protein targeting validation, indicating decreased Pin1 expression in osteoarthritis. Furthermore, by using lentiviral interference / overexpression to modulate Pin1 protein expression levels in chondrocytes and detecting chondrocyte viability using CCK8 assays, it was concluded that Pin1 plays a crucial role in maintaining chondrocyte growth. In clinical samples of human knee replacement surgery, articular cartilage wear and degeneration in the weight-bearing area were severe, and Pin1 protein levels were significantly reduced. Osteoarthritis was more easily induced in a mouse model with cartilage-specific Pin1 knockout. Therefore, Pin1 protein can serve as an important biomarker associated with osteoarthritis. Using reagents corresponding to Pin1 protein, its expression level can be detected, thereby achieving the purpose of diagnosing osteoarthritis and / or assessing the risk of developing osteoarthritis. Furthermore, detecting the expression level of Pin1 protein can be used to screen or prepare drugs for treating osteoarthritis, and simultaneously evaluate the efficacy and quality of these drugs. Therefore, the discovery of a novel biomarker related to osteoarthritis—Pin1 protein—has significant biological and pharmacological implications.

[0028] Furthermore, the inventors of this invention, based on Western blotting and enzyme-linked immunosorbent assay (ELISA) of Pin1 in rat serum, found that Pin1 was significantly downregulated in the osteoarthritis model group; after treatment with Rongjin Niantong Decoction, the expression level of Pin1 protein was significantly upregulated. This suggests that Rongjin Niantong Decoction can promote the expression of Pin1 in rat knee cartilage and delay cartilage degeneration. Pin1 protein is a novel target of Rongjin Niantong Decoction in treating osteoarthritis and can serve as a new biomarker related to the pharmacodynamic function of Rongjin Niantong Decoction in treating osteoarthritis. The discovery of the new target Pin1 protein makes the pharmacodynamic function research of Rongjin Niantong Decoction more comprehensive and richer, laying a foundation for further research on the specific mechanism of Rongjin Niantong Decoction in treating osteoarthritis and the identification of biomarkers. Attached Figure Description

[0029] Figure 1 This is a flowchart of the experimental and analytical process for TMT proteomics in Example 1.

[0030] Figure 2 This is a diagram showing the results of Example 1 in identifying Pin1 as a potential target for osteoarthritis; where, Figure 2 A represents a Wayne analysis of osteoarthritis disease targets, TMT proteomics, and protein targeting validation. Figure 2 B represents the expression of Pin1 protein in the normal group and osteoarthritis group during TMT proteomics and protein targeting validation.

[0031] Figure 3 This is a diagram showing the effect of Pin1 interference on chondrocytes observed in Example 2.

[0032] Figure 4 This is a diagram showing the effect of Pin1 interference on chondrocytes as observed under a microscope in Example 2.

[0033] Figure 5 This is a diagram showing the effect of Pin1 interference on chondrocyte growth and viability in Example 2.

[0034] Figure 6 This is a diagram illustrating the effect of Pin1 overexpression on chondrocyte growth viability in Example 2; where... Figure 6 A and Figure 6 B represents observation under a fluorescence microscope, where... Figure 6 A represents the overexpression control group. Figure 6 Group B represents the overexpression group; Figure 6 C and Figure 6 D represents observation under a regular microscope, where Figure 6 C represents the overexpression control group. Figure 6 D represents the overexpression group; Figure 6 E represents the CCK8 test result.

[0035] Figure 7 This is a 10× image showing the HE staining results of the knee joint cartilage of three individuals in Example 3.

[0036] Figure 8 This is a diagram showing the expression of Pin1 protein in the knee cartilage of three individuals in Example 1. Figure 8 A is the WB result image. Figure 8 B is Figure 8 A statistical chart, * P<0.05.

[0037] Figure 9 This is an immunofluorescence image (40×) of Pin1 expression in the knee cartilage of transgenic mice in Example 4.

[0038] Figure 10The image shows the HE and Safranin-Fix Green staining results of the knee joint cartilage of transgenic mice in Example 4 (10×). In the image, AD shows HE staining and EH shows Safranin-Fix Green staining.

[0039] Figure 11 This is a diagram showing the expression of Pin1 in articular cartilage and serum in Example 5; where, Figure 11 A represents the expression of Pin1 protein in articular cartilage; Figure 11 B is Figure 11 A statistical chart; Figure 11 C represents the expression level of Pin1 in serum. Compared with the control group: ▲▲ P < 0.01, ▲ P < 0.05; compared with the model group: ★★ P < 0.01, ★ P < 0.05. Detailed Implementation

[0040] The present invention will now be described in detail with reference to the accompanying drawings and embodiments:

[0041] Example 1: Identifying Pin1 as a potential target (i.e., biomarker) associated with osteoarthritis.

[0042] Experimental steps:

[0043] Acquisition of disease targets for osteoarthritis

[0044] Using "osteoarthritis" as the keyword, we downloaded files related to osteoarthritis research from the GeneCard database, annotated and grouped the downloaded files, and finally performed differential analysis on the data to screen out targets for OA.

[0045] Establishment of a knee osteoarthritis model

[0046] Thirty SPF-grade 2-month-old male SD rats, weighing 230±20g, were randomly divided into a control group (n=10) and a model group (n=20) using a random number table after one week of acclimatization. The model group was further divided into a model group (n=10) and a Rongjin Niantong Fang group (n=10). All rats were fasted for 12 hours preoperatively but allowed free access to water. The control group underwent sham surgery, where only the skin of both knee joints was incised without removing the menisci; the incision was sutured and then disinfected. The model group underwent a modified Hulth method: a medial approach was made to both knee joints of the hind legs, the medial collateral ligament was severed, the medial meniscus was removed, the anterior cruciate ligament was severed, and the incision was sutured and then disinfected. Postoperatively, rats received intramuscular injections of 200,000 U / day of penicillin for three consecutive days.

[0047] Administration and sampling of rats

[0048] One week after the rat model was established, they basically recovered to their normal state. The intervention methods for each group are as follows:

[0049] Control group: 1 mL of physiological saline was administered by gavage to rats weighing 250 g once daily.

[0050] Model group: 1 mL of physiological saline was administered by gavage to rats weighing 250 g once daily.

[0051] Rongjin Niantong Formula Group: The amount of concentrated water extract of Rongjin Niantong Formula administered to rats was calculated based on the conversion of human and rat body surface area, i.e., rat dose = human dose × 6.3, once daily.

[0052] Rats were weighed weekly, and the dosage was adjusted based on changes in body weight. The intervention was continued for 12 weeks.

[0053] After 12 weeks of drug intervention, rats were fasted but allowed free access to water for 12 hours. After anesthesia with isoflurane, knee joint cartilage was harvested, washed three times with physiological saline, placed in 1.5 mL EP tubes, and quickly stored in liquid nitrogen before being transferred to a -80°C freezer for storage.

[0054] TMT Proteomics

[0055] The experimental workflow for TMT proteomics mainly includes: tissue protein extraction, FASP digestion, isotope labeling, peptide mixing and analysis, protein identification, and bioinformatics analysis. See details below. Figure 1 .

[0056] (1) Sample mixing and biological replication: Cartilage from the bilateral tibial plateaus of 3 rats in each group is mixed to form a biological sample, that is, 9 rats are needed for 3 biological replicates in each group.

[0057] (2) Tissue protein extraction and quantification: Tissue proteins were extracted using TCA / acetone precipitation and SDT lysis. Specifically, an appropriate amount of sample was ground into bone powder in a mortar containing liquid nitrogen. Five volumes of TCA / acetone (1:9) were added, mixed thoroughly, and the mixture was allowed to precipitate overnight at -20°C. The supernatant was removed by centrifugation, and the precipitate was washed with pre-cooled acetone and dried. Approximately 30 mg of the dried precipitate was weighed, and 30 volumes (m / v) of lysis buffer were added. After mixing, the mixture was incubated in a boiling water bath for 5 min, followed by ultrasonic disruption and then incubation in a boiling water bath for 15 min. The mixture was then centrifuged at high speed (14000g) for 30 min. Protein quantification was performed using the BCA method. After aliquoting, the samples were stored at -80°C. All protein samples were analyzed by 12.5% ​​SDS-PAGE electrophoresis (constant current 14mA, 90 min), with 20 μg transferred to each well.

[0058] (3) FASP digestion: The extracted protein was digested using the FASP method, i.e., 30 μL of each sample was taken, DTT was added to a final concentration of 100 mM, incubated at 100℃ for 5 min, cooled to room temperature, and then 200 μL of UA buffer was added and mixed. The mixture was centrifuged at 14000 rpm for 15 min using a 10 KD ultrafiltration tube (repeated once), and 100 μL of 100 mM IAA buffer was added to each tube. The mixture was shaken at 600 rpm for 1 min, incubated at room temperature in the dark for 30 min, and then centrifuged at 14000 rpm for 15 min. The mixture was washed twice with 100 μL of UA buffer and twice with 100 μL of 100 mM TEAB buffer. 40 μL of trypsin (4 μg of trypsin dissolved in 40 μL of 100 mM TEAB buffer) was added, mixed, and then replaced with a new collection tube. The mixture was digested at 37℃ for 16-18 h. After enzymatic hydrolysis, the sample was centrifuged at 14000g for 15 min, washed once with 40 μL of 10 mM TEAB buffer, and the combined filtrate was collected for determination of peptide content (OD280).

[0059] (4) TMT labeling: Take 100 μg of peptide from each sample and follow the TMT kit operation steps.

[0060] (5) RP fractionation of peptides: Peptides were labeled, lyophilized, and mixed in equal volumes. 100 μg of the peptide mixture was diluted with 300 μL of 0.1% TFA, separated by reversed-phase high-pH chromatography, and the eluent fractions were collected. Each fraction was lyophilized and reconstituted with 12 μL of 0.1% formic acid. The absorbance of each peptide fraction was measured at 280 nm, and the concentration of each fraction was calculated. The reversed-phase high-pH chromatography conditions were: Agilent 1100 HPLC, Agilent Zorbax Extend-C18 narrow-diameter column (2.1 × 150 mm, 5 pm).

[0061] (6) Polypeptide separation: Polypeptides were separated after proteolytic digestion using nano-liquid chromatography-high-resolution mass spectrometry. The column was equilibrated with 0.1% formic acid solution for 30 min, and eluted according to the separation conditions in Table 1:

[0062] Table 1. Liquid Chromatography Elution Procedure

[0063]

[0064] Note: Mobile phase A is 0.1% formic acid; mobile phase B is 0.1% formic acid-acetonitrile aqueous solution (acetonitrile volume percentage is 84%).

[0065] The high-resolution Q-Exactive mass spectrometer was used in positive ion mode, with a scan range of 300-1800 m / z. Ten secondary fragment spectra were acquired after each full scan. The MS resolution was set to 70,000, and the MS / MS resolution was set to 17,500. The collision energy was 30 eV.

[0066] (7) Protein identification and relative quantitative analysis:

[0067] Mass spectrometry data were analyzed using Masscot 2.2 and Proteome Discoverer 1.4 for database identification and relative quantification. Specific parameter settings are detailed in Table 2. Protein quantification was performed based on the median quantification value of the unique peptide in the protein, and data correction was applied based on the median quantification value.

[0068] Table 2. Parameter settings for Masscot 2.2 and Proteome Discoverer 1.4

[0069]

[0070] Protein targeting validation

[0071] Liquid chromatography: Easy nLC 1200 system (Thermo Scientific)

[0072] Mass spectrometry: Q-Exactive HF (Thermo Scientific)

[0073] Trapping column: Homemade column (100μm*50mm, 5μm-C18)

[0074] Analytical column: Homemade tip column (75μm*200mm, 3μm-C18)

[0075] Proteolytic digestion:

[0076] (1) Sample preparation and protein extraction are the same as FASP digestion in TMT proteomics.

[0077] For each sample, approximately 200 μg of protein was taken, and DTT was added to a final concentration of 100 mM. The mixture was then incubated in a boiling water bath for 15 min. After cooling to room temperature, 200 μL of UA buffer (8 M Urea, 150 mM Tris-HCl, pH 8.0) was added, mixed, and transferred to a 10 kDa ultrafiltration tube. The tube was centrifuged at 14000 g for 30 min. Next, 200 μL of LUA buffer was added, and the tube was centrifuged at 14000 g for 30 min. The filtrate was discarded. 100 μL of 50 mM IAA was added, and the mixture was vortexed at 600 rpm for 1 min, incubated at room temperature in the dark for 30 min, and centrifuged at 14000 g for 20 min. The process was repeated three times: adding 100 μL of 50 mM IAA buffer and centrifuging at 14000 g for 20 min. Centrifuge at 14000g for 20 min in NH4HCO3 buffer, repeat twice; add 40 μL NH4HCO3 buffer (containing Trypsin, enzyme ratio 1:50), shake at 600 rpm for 1 min, incubate at 37℃ for 16 h; replace with a new collection tube, centrifuge at 14000g for 15 min. Add 40 μL 50 mM NH4HCO3 buffer, centrifuge at 14000g for 30 min, collect the filtrate; desalt and lyophilize the enzymatically hydrolyzed peptides, then reconstitute with 0.1% FA, and determine the peptide concentration by OD280.

[0078] (2) Liquid Chromatography Analysis

[0079] Based on the preliminary experimental results, the target peptides of the identified target proteins were subjected to PRM quantitative analysis. Peptide information suitable for PRM analysis was imported into the Xcalibur software for PRM method setup. Approximately 1 μg of peptide was taken from each sample and incorporated with 20 fmol of standard peptide (PRTC: ASEFDSAIAQDK) for detection. Chromatographic separation was performed using an HPLC system; the elution conditions are shown in Table 3.

[0080] Table 3. Liquid Chromatography Elution Procedures for Protein Target Validation

[0081]

[0082] Note: Mobile phase A is 0.1% formic acid; mobile phase B is 0.1% formic acid-acetonitrile aqueous solution (acetonitrile volume percentage is 84%).

[0083] (3) High-resolution mass spectrometry (PRM / MS) analysis

[0084] Samples separated by high-performance liquid chromatography (HPLC) were analyzed by PRM mass spectrometry using a Q-Exactive HF mass spectrometer (Thermo Scientific). Analysis time: 60 min; detection mode: positive ion; primary mass spectrometry scan range: 300-1800 m / z; mass spectrometry resolution: 60000 (m / z 200); AGC target: 3e6; maximum IT: 200 ms. After each primary MS scan (full MSscan), 20 PRM scans (MS2 scans) were acquired according to the inclusion list. Isolation window: 1.6Th; mass spectrometry resolution: 30000 (m / z 200); AGC target: 3e6; maximum IT: 120 ms; MS2 activation type: HCD; normalized collision energy: 27. PRM detection was performed on the samples, and the raw PRM files were finally analyzed using Skyline 3.5.0 software.

[0085] Experimental results:

[0086] Wayne's analysis of osteoarthritis disease targets, TMT proteomics, and protein targeting validation revealed that among the six proteins with common intersection, Pin1 showed a consistent trend in both TMT proteomics and protein targeting validation, indicating that Pin1 expression is decreased in osteoarthritis. Figure 2 ).

[0087] Example 2: Pin1 is an important protein for maintaining chondrocyte growth.

[0088] Experimental steps:

[0089] Pin1 lentiviral interference / overexpression in chondrocytes

[0090] When the F0 generation cells reach 70%-80% confluence, they can be passaged for subsequent experiments. This experiment uses F1 generation chondrocytes; therefore, cells reaching 70%-80% confluence are used. The specific experimental steps are as follows:

[0091] (1) Following the cell passage procedure, F0 generation cells were digested and counted.

[0092] (2) Dilute the cells to the required concentration according to the required total cell volume and number.

[0093] (3) After mixing, plate the cells. ① 96-well plate method: Add 100 μL of cell suspension to each well using a pipette, with 5000 cells per well. ② 6-well plate method: 60,000 cells per well, mix and spread evenly using a pipette.

[0094] (4) After about 16 hours of plating, dilute the virus according to the required MOI value (the required virus volume V = (MOI × number of cells) / virus titer), and prepare it in EP tubes. Use lentivirus to interfere with cells by changing the medium.

[0095] (5) Starting from the time the virus was added, replace each well with fresh 10% FBS DMEM medium at 12h.

[0096] (6) Starting from the time of virus addition, photograph the fluorescence and state of cells 96 hours after lentivirus interference.

[0097] CCK8 assay for chondrocyte viability

[0098] (1) According to experimental requirements, seed the cell suspension (100 μL / well) into 96-well plates, and fill the blank wells with PBS or blank culture medium. When the cell intervention is completed, the viability of CCK8 cells can be detected.

[0099] (2) To ensure the accuracy of sample addition, dilute the CCK8 culture medium with blank culture medium according to the number of wells to be tested (add 10 μL of CCK8 solution to every 100 μL of blank culture medium), and then add 100 μL of diluted CCK8 solution to each well.

[0100] (3) Place the culture plate in an incubator and incubate for 2 hours.

[0101] (4) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 450 nm.

[0102] Experimental results:

[0103] from Figure 3 and Figure 4 The results show that lentivirus interference with Pin1 protein expression in chondrocytes leads to a change in chondrocyte morphology from round to elongated, and a decrease in chondrocyte number, with the most significant changes observed in interference 2. Simultaneously, it significantly inhibits chondrocyte viability ( ) from 48 to 120 hours. Figure 5 Conversely, lentiviral overexpression of Pin1 protein in chondrocytes significantly promoted chondrocyte growth and viability. Figure 6 ).

[0104] Example 3: Clinical Trial

[0105] 1 clinical sample

[0106] Three patients who met the diagnostic criteria for KOA in the "Guidelines for the Diagnosis and Treatment of Osteoarthritis (2021 Edition)" and underwent knee replacement surgery were selected. The tibial plateau was used after knee replacement surgery. Smooth cartilage from the non-weight-bearing area of ​​the articular surface was used as the control group, while significantly worn cartilage from the weight-bearing area of ​​the articular surface was used as the experimental group.

[0107] 2 Experimental Methods

[0108] 2.1 HE staining to observe pathological changes in knee articular cartilage

[0109] The collected cartilage from knee replacement surgery was stained with hematoxylin-eosin (HE) and observed for pathological changes under a microscope.

[0110] ① Fix the cartilage tissue in 4% decalcification solution for 24-48 hours, rinse off the 4% paraformaldehyde fixative solution with running water, and then place it in 10% EDTA solution for decalcification. Change the decalcification solution every other day until it becomes soft like soft tissue.

[0111] ② After gradient dehydration, embedding, sectioning, and dewaxing, the tissue is stained with hematoxylin for 60-90 seconds (adjust according to the staining solution), rinsed slowly with running water for 5 minutes, and observed under a microscope. The hematoxylin staining time can be adjusted as needed. ② Differentiation solution is applied for 3 seconds, followed by rinsing slowly with running water for 10 minutes. ③ Eosin is applied for 3-4 seconds, followed by rinsing with running water for 5 minutes. The staining is observed under a microscope, and the eosin staining time can be adjusted as needed. After drying, the tissue is photographed under a microscope.

[0112] 2.2 Western Blot (WB) assay to detect the expression of Pin1 protein in knee joint cartilage tissue.

[0113] ① Cartilage tissue was ground into powder in liquid nitrogen, then RIPA lysis buffer was added and homogenized for 5 minutes, followed by lysis on ice for 1 hour. ② The mixture was centrifuged at 14000 rpm for 30 minutes, and the supernatant protein solution was collected. ③ The total protein concentration was determined using the BCA protein quantification method. ④ After protein denaturation, SDS-PAGE gel electrophoresis, membrane transfer, blocking, incubation with primary and secondary antibodies, and finally development by electrochemiluminescence (ECL) imaging. Images were processed using ImageLab software for semi-quantitative analysis.

[0114] 2.3 Statistical Analysis

[0115] All data were analyzed using IBM SPSS Statistics 27 software. Two independent samples t-tests were used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0116] 3 Results

[0117] 3.1 HE staining showed significant cartilage tissue lesions in osteoarthritis.

[0118] HE staining results showed that the control group had smooth, intact cartilage surfaces and homogeneous extracellular matrix; compared with the control group, the experimental group had rough cartilage surfaces, thinner cartilage, and reduced extracellular matrix (e.g., Figure 7 (As shown).

[0119] 3.2WB results showed decreased Pin1 protein levels in osteoarthritis cartilage tissue.

[0120] Compared with the control group, the experimental group showed a significant decrease in Pin1 protein content in cartilage (P < 0.05), suggesting that Pin1 is a marker of cartilage degeneration in osteoarthritis (e.g., Figure 8 (As shown).

[0121] Example 4: Transgenic Mouse Experiment

[0122] 1. Transgenic mouse information

[0123] SPF-grade C57BL / 6J-Pin1 mice and Col2-Cre mice were purchased from Cyagen Model Organisms Research Center (Taicang) Co., Ltd., Animal Qualification Certificate No.: NO.202129237. All experimental procedures were strictly in accordance with the relevant regulations of the Medical Ethics Committee of Fujian University of Traditional Chinese Medicine. Ethics Review No.: FJTCM IACUC2021010. Pin1 mice were obtained after breeding these two mouse species. flox / flox (Pin1 + / + ) and Col2-Cre; Pin1 flox / flox (Pin1 - / - Two mouse genotypes were used for experimental research.

[0124] 2 Experimental Methods

[0125] 2.1 Establishment and Material Collection of Transgenic Mouse Osteoarthritis Model

[0126] 8-week-old male Pin1 + / + and Pin1 - / - Ten mice of each type were selected, and the selected Pin1 mice were randomly assigned using a random number method. + / + Type and Pin1 - / - The two types of mice are divided into Pin1 and Pin2. + / + Sham surgery group (5 animals), Pin1 + / + Modeling kit (5 pieces), Pin 1 - / - Sham surgery group (5 animals), Pin1 - / -Model group (n=5). A modified Hulth technique was used to establish a KOA model. Mice were fasted for 12 hours preoperatively but allowed free access to water, and anesthetized with isoflurane (1.5%–2.0%). After adequate anesthesia, the surgical area on the medial side of both knee joints was shaved and prepared, and disinfected with iodine-soaked cotton balls. A 0.5cm incision was then made on the medial side of the knee joint, and the muscle tissue was bluntly dissected. In the model group, the medial collateral ligament was severed to expose the knee joint cavity, and the patellar ligament was laterally rotated. Subsequently, the anterior cruciate ligament was severed, and the medial meniscus was removed. Care was taken to avoid damaging the articular surfaces throughout the procedure. Finally, the joint was irrigated with saline and iodine, and the joint capsule, medial knee muscles, and surgical incision were sutured layer by layer. In the sham surgery group, only the muscle tissue was incised and dissected, and then the incision was sutured layer by layer. Postoperatively, the mice were closely monitored. After recovery, each mouse was given an intramuscular injection of 6000U of penicillin sodium for 3 consecutive days to prevent infection.

[0127] Eight weeks after modeling, the patients were anesthetized with isoflurane (1.5%–2.0%). Both knee joints were bluntly dissected on ice, and surrounding attached muscles and soft tissues were removed, taking care to preserve osteophytes. The tissues were fixed for 24 hours in 4% paraformaldehyde fixative solution approximately 10 times the volume of the tissue block. The 4% paraformaldehyde fixative solution was rinsed off with running water, and the tissues were placed in 10% EDTA solution for decalcification. The decalcification solution was changed every other day until the tissues were soft like soft tissue. The tissues underwent gradient dehydration, embedding, sectioning, and dewaxing / hydration.

[0128] 2.1 Immunofluorescence staining of the knee joint of transgenic mice

[0129] After dewaxing, the paraffin sections were placed in a beaker containing sodium citrate antigen retrieval solution boiled at 100°C for antigen retrieval. The beaker was placed in a water bath at a temperature above 95°C for 15 minutes. After retrieval, the sections were washed three times with 1×PBS for 5 minutes each time. The tissue location was delineated approximately 0.5 cm away with an immunohistochemical pen, and the membrane was perforated with 0.5% Triton for 20 minutes. The sections were then washed three times with 1×PBS for 5 minutes each time, blocked with 5% BSA for 30 minutes, and primary antibody was prepared according to the specified ratio. The sections were incubated overnight at 4°C. The sections were washed three times with 1×PBS for 5 minutes each time, and secondary antibody was prepared according to the specified ratio and species. The sections were incubated at room temperature in the dark for 1 hour, and then washed three times with 1×PBS in the dark for 5 minutes each time. Finally, a DAPI-containing anti-fluorescence quencher was added and the sections were mounted in the dark. The sections were then observed and photographed under an inverted fluorescence microscope.

[0130] 2.1 HE and Safranin-Fix-Green staining to observe pathological changes in knee articular cartilage

[0131] ① HE staining: After dewaxing paraffin sections, stain with hematoxylin for 60-90 seconds (adjust according to the staining solution), rinse slowly with running water for 5 minutes, observe the staining under a microscope, and adjust the hematoxylin staining time as needed; differentiate with differentiation solution for 3 seconds, rinse slowly with running water for 10 minutes. Apply eosin for 3-4 seconds, rinse with running water for 5 minutes, observe the staining under a microscope, and adjust the eosin staining time as needed. After drying, photograph the sections under a microscope.

[0132] ② Safranin-Fixed Green Staining: After dewaxing paraffin sections, mix safranin-fixed green solution A (A1: Weigert A solution) and solution B (A2: Weigert B solution) in a 1:1 ratio to prepare Weigert iron-hematoxylin staining solution (prepare fresh before use; it loses its staining ability after 24 hours, protect from light). Apply the solution to the tissue for approximately 15-20 seconds. Discard the staining solution and then soak the tissue in pure water for 5 minutes. Apply Fast Green staining solution to each tissue section for approximately 5 seconds. After soaking in pure water for 5 minutes, adjust the staining time according to the color of Fast Green under the microscope. Apply Safranin staining solution to each tissue section for approximately 5-10 seconds. After soaking in pure water for 5 minutes, adjust the staining time according to the color of Safranin under the microscope. Air dry at room temperature, mount with neutral resin, and photograph under a microscope.

[0133] 3 Results

[0134] 3.1 Pin1 knockout in the knee cartilage of transgenic mice showed good results.

[0135] Figure 9 Immunofluorescence results of Pin1 in the knee joint of transgenic mice showed that: Pin1 without knockout + / + In the group, Pin1 protein was normally expressed in the cartilage layer, but in the Pin1 knockout group... - / - In the group, the fluorescence intensity of Pin1 was significantly reduced, indicating that the knockout effect of Pin1 in the knee cartilage of transgenic mice was good.

[0136] 3.2 Pin1 knockout exacerbates the progression of osteoarthritis

[0137] Figure 10 AD is the HE staining result of the knee joint of transgenic mice: from Figure 10 As can be seen from A and 10B, it is related to Pin1 + / + Compared to the previous group, Pin1 - / - In this group, the knee joint cartilage layer was thinned, chondrocytes were arranged disorderedly, and the cartilage surface was severely worn; from Figure 10 As can be seen from C and 10D, it is related to Pin1 + / + Compared to the previous group, Pin1 - / - The cartilage layer in the group not only became thinner, but also showed extremely severe wear and defects. Figure 10 EH represents the results of Safranin-Fixgreen staining of the knee joint in transgenic mice: From Figure 10As can be seen from E and 10F, it is related to Pin1 + / + Compared to the previous group, Pin1 - / - In this group, the knee joint cartilage layer was rough, and the safranin staining of the cartilage layer was severe; similarly, from Figure 10 As can be seen from G and 10H, it is related to Pin1 + / + Compared to the previous group, Pin1 - / - The cartilage in the group was not obvious, and extremely severe deformation was observed. The above staining results indicate that Pin1 is an important protein for maintaining the normal function of knee joint cartilage, and knocking out Pin1 in knee joint cartilage will exacerbate cartilage wear and degeneration under pathological conditions.

[0138] Example 5: Pin1 is the target point for Rongjin Niantong Formula in treating osteoarthritis.

[0139] Experimental steps:

[0140] Western blot

[0141] (1) Select a glass plate and cover plate of 1.0 mm or 1.5 mm according to the experimental plan, and wash with pure water until there are no water droplets on the glass plate and no water sticks to the wall.

[0142] (2) Clamp the glass plate and test for leaks with pure water. The liquid level should drop by no more than 2 mm within 5 minutes. If the liquid level drops significantly, the glass plate can be disassembled and reinstalled.

[0143] (3) Pour out the pure water from the glass plate, tilt it, and wash away the remaining water with filter paper.

[0144] (4) Prepare an appropriate volume of separating gel according to the gel preparation kit. After adding the coagulant, mix well and immediately pour it into the top of the crossbeam of the gel preparation rack.

[0145] (5) Add anhydrous ethanol and flatten it. Let it stand for about 30 minutes, or observe the remaining separating gel solution in the tube until the remaining separating gel solution solidifies.

[0146] (6) Discard the anhydrous ethanol and blot dry with filter paper. Select a 10-well or 15-well comb according to the sample loading volume required for the experiment and prepare it.

[0147] (7) Prepare the upper stacking gel according to the instructions of the gel preparation kit, add it to each glass plate, and insert the comb (pay attention to observe when inserting the comb to avoid pressing air bubbles inside).

[0148] (8) Let stand for 30 minutes, or observe the remaining separating gel solution in the tube until the remaining separating gel solution solidifies (the prepared gel can be soaked in electrophoresis buffer and used the next day, and stored at 4°C).

[0149] (9) Install the prepared gel into the electrophoresis clamp (make sure it is securely clamped to avoid leakage) and place it in the electrophoresis tank. Add new electrophoresis buffer to the inner tank, and you can use old electrophoresis buffer for the outer tank (before using old electrophoresis buffer, shake the electrophoresis solution; if there is white flocculent material, it cannot be used).

[0150] (10) Thaw the sample on ice and denature it at 100°C for 5 min.

[0151] (11) Slowly pull out the comb with both hands vertically upwards, and blow each hole with a 200μL pipette to blow out any remaining glue or other residues.

[0152] (12) Load the sample according to the experimental plan, leaving space on both sides for standard protein (3 μL / well), and fill the blank wells with 1× loading buffer.

[0153] (13) Set the power supply conditions for the electrophoresis apparatus (① 40V, 20min; ② 80V, 30min; ③ 120V, 55min) to allow the sample to enter the stacking gel for concentration and separation. (After turning on the power, observe whether bubbles are generated. The presence of bubbles indicates normal operation. Observe whether the bromophenol blue at the sample position is moving downwards. If the bromophenol blue moves upwards, immediately turn off the power and check whether the electrodes are inserted in reverse to avoid sample loss.)

[0154] (14) Prepare a new transfer solution and pre-cool the new and old transfer solutions in a -20°C refrigerator.

[0155] (15) Cut a 0.22μm PVDF membrane of appropriate size and immerse it in methanol for 5 min to activate it.

[0156] (16) After electrophoresis, the glass plate is removed and immersed in the recovered pre-cooled transfer solution.

[0157] (17) Divide the gel into two parts: high molecular weight (greater than 70 KD) and low molecular weight (less than 70 KD), and transfer them separately. The sandwich sandwich order is: black side - mesh support pad - filter paper - gel - membrane - filter paper - mesh support - white side. There should be no air bubbles between the membrane and the gel. After sandwiching, place them in the transfer tank with black side to black side and white side to red side. Transfer the high molecular weight gel on ice for 70 min and the low molecular weight gel on ice for 35 min.

[0158] (18) Based on the molecular weight of the target protein, cut the membrane and place it in an incubator and wash it once in TBST.

[0159] (19) Discard TBST, absorb dry, add 5 mL of blocking solution to each well, and seal on a shaker at room temperature for 1 h.

[0160] (20) Wash once with TBST, dry, add primary antibody, and incubate overnight at 4°C.

[0161] (21) Recover the primary antibody. Wash each well with TBST 3 times, 5 min each time.

[0162] (22) Add the corresponding secondary antibody and incubate at room temperature for 1 hour.

[0163] (23) Clean each well with TBST 3 times, 5 minutes each time.

[0164] (24) Prepare the developing solution (1:1 ratio, prepare fresh each time) and develop it on a chemiluminescence developer.

[0165] Enzyme-linked immunosorbent assay (ELISA) of Pin1 in rat serum

[0166] Preparation of rat serum: After 12 weeks of drug intervention, rats were fasted but allowed to drink water for 12 hours. After isoflurane anesthesia, about 4-5 mL of blood was collected from the abdominal aorta of rats using an inert separation gel coagulation tube (yellow cap). One tube was collected per rat, left to stand at room temperature for 2-4 hours, centrifuged at 3500 rpm for 15 minutes at 4℃, aliquoted into 100 μL tubes, and stored at -80℃.

[0167] (1) Take 8 samples of rat serum from each of the control group, model group and Rongjin Niantong Formula group described in Example 1 as test samples, thaw them on ice, and perform enzyme-linked immunosorbent assay (ELISA) on rat serum for Pin1.

[0168] (2) Following the instructions for use of the Pin1 enzyme-linked immunosorbent assay kit, dilute the standard to 480 pg / mL, 240 pg / mL, 120 pg / mL, 60 pg / mL and 30 pg / mL.

[0169] (3) Sample addition: Set up blank wells (blank control wells do not contain sample or enzyme-labeled reagent, all other steps are the same), standard wells, and sample wells. Accurately add 50 μL of standard to the enzyme-labeled plate. Add 40 μL of sample diluent to the sample wells, and then add 10 μL of sample to the sample wells (the final sample dilution is 5 times). When adding the sample, place it at the bottom of the well, avoiding touching the well wall as much as possible, and gently shake to mix.

[0170] (4) Incubation: After sealing the plate with sealing film, incubate at 37°C for 30 min.

[0171] (5) Solution preparation: Dilute the concentrated washing solution with distilled water to 1×.

[0172] (6) Washing: Carefully peel off the sealing film, discard the liquid, shake dry, fill each hole with washing liquid, let stand for 30 seconds and then discard. Repeat this 5 times, pat dry (placing a lint-free paper underneath and inverting to absorb the dryness).

[0173] (7) Add enzyme: Add 50 μL of enzyme labeling reagent to each well, except for blank wells.

[0174] (8) Incubation: The procedure is the same as in 4.

[0175] (9) Washing: Same as 6.

[0176] (10) Color development: Add 50 μL of color developer A solution to each well, then add 50 μL of color developer B solution, gently shake to mix, and develop color at 37℃ in the dark for 10 min.

[0177] (11) Termination: Add 50 μL of stop solution to each well to terminate the reaction (at this time, the blue color will immediately turn yellow).

[0178] (12) Measurement: Zero the instrument with the blank well and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0179] Experimental results:

[0180] from Figure 11 As shown in A and 11B, Pin1 was significantly downregulated in the osteoarthritis model group; after treatment with Rongjin Niantong Decoction, the expression level of Pin1 protein was significantly upregulated, suggesting that Rongjin Niantong Decoction can promote the expression of Pin1 in the knee cartilage of rats. Figure 11 As shown in Figure C, the serum Pin1 protein level in the osteoarthritis model group showed a decreasing trend, and the intervention of Rongjin Niantong Decoction could upregulate the expression of Pin1 protein in the serum. Therefore, the treatment of osteoarthritis with Rongjin Niantong Decoction may be related to Pin1.

[0181] The embodiments of this example have been described above. However, this example is not limited to the specific implementation methods described above. The specific implementation methods described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms based on the guidance of this example, and all of them are within the protection scope of this example.

Claims

1. The use of a biomarker in the preparation of products for diagnosing osteoarthritis and / or assessing the risk of developing osteoarthritis, characterized in that: The biomarker is the Pin1 protein.

2. The application according to claim 1, characterized in that: The products include chips or reagent kits.

3. The application of a biomarker in screening or preparing drugs for treating osteoarthritis, characterized in that: The biomarker is the Pin1 protein.

4. The application of a biomarker in the preparation of a reagent for evaluating the quality of drugs for the treatment or relief of osteoarthritis, characterized in that: The biomarker is the Pin1 protein.

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