Application of Sirt1 in preparation of product for diagnosis, prognosis or treatment of steroid-induced femoral head necrosis

Abstract

The invention relates to application of Sirt1 in preparation of products for diagnosis, prognosis or treatment of steroid-induced femoral head necrosis, and belongs to the technical field of biotechnology and medicine. It is found that Sirt1 participates in dexamethasone-induced osteoblast ferroptosis, differential expression of Sirt1 can significantly affect the degree of dexamethasone-induced osteoblast ferroptosis, and occurrence of the steroid-induced femoral head necrosis phenomenon is prompted. The invention provides application of Sirt1 as a marker molecule for diagnosing steroid-induced femoral head necrosis in preparation of a reagent for diagnosing or screening steroid-induced femoral head necrosis, and provides application of an expression promoter of Sirt1 in preparation of a medicine for treating steroid-induced femoral head necrosis.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and medical technology, and specifically relates to the application of Sirt1 in preparing products for diagnosing, prognosing or treating hormone-induced femoral head necrosis. Background Art

[0002] Steroid-induced osteonecrosis of femoral head (SONFH) is a serious bone disease caused by long-term or excessive use of hormone drugs (such as glucocorticoids). It is common in young and middle-aged people aged 30 to 50 years old, mainly affecting the blood supply of the femoral head, leading to the death of bone matrix cells, trabecular fracture, and may eventually cause femoral head collapse and dysfunction. It is one of the main diseases causing loss of working ability. Osteoblasts are the main functional cells of bone formation, responsible for the synthesis, secretion and mineralization of bone matrix, and play an important role in bone formation and injury repair. The pathogenesis of SONFH is complex. Studies have shown that glucocorticoids can induce the occurrence of osteoblast ferroptosis, inhibit osteogenic function, and thus affect the progression of steroid-induced osteonecrosis of femoral head. Dexamethasone (Dex) is a synthetic long-acting glucocorticoid drug that mainly has anti-inflammatory and immunosuppressive effects. Long-term use can cause avascular necrosis of the femoral head. At present, the diagnosis of SONFH is mainly based on imaging diagnosis, but this type of diagnostic method is expensive and has poor portability, which is not conducive to the popularization of disease screening and detection methods. With the development of molecular biology, biomarkers are often used for the prevention, diagnosis and treatment of diseases. Sirt1, as a potential biomarker, has achieved certain research results in the diagnosis of rheumatoid arthritis and leukemia, but its application in the field of femoral head necrosis is still in its infancy. Therefore, it is of great clinical significance to apply Sirt1 to the diagnosis and prognosis of hormone-induced femoral head necrosis and the development of therapeutic drugs. Summary of the invention

[0003] In response to the problems existing in the prior art, the present invention provides Sirt1 for preparing products for diagnosing, prognosing or treating hormonal femoral head necrosis, Sirt1 for preparing products for early and prognostic diagnosis of hormonal femoral head necrosis, and Sirt1 expression promoter for preparing drugs for treating hormonal femoral head necrosis, thereby achieving the purpose of diagnosing, prognosing and treating hormonal femoral head necrosis.

[0004] The present invention provides an application of Sirt1 in preparing a product for diagnosing, prognosing or treating hormone-induced femoral head necrosis. The application is that Sirt1 is used as a diagnostic marker. When the product detects an increased expression level of Sirt1, it indicates that the prognosis of hormone-induced femoral head necrosis is good.

[0005] Furthermore, the product includes a chip, a kit or a nucleic acid membrane strip capable of detecting Sirt1 expression.

[0006] Furthermore, the chip is a gene chip or a protein chip. The gene chip is an oligonucleotide probe targeting the Sirt1 gene for detecting the transcription level of the Sirt1 gene, and the protein chip is a specific binder of the Sirt1 protein.

[0007] The kit is a gene detection kit or a protein detection kit. The gene detection kit is a reagent or a chip for detecting the transcription level of Sirt1 gene, and the protein detection kit is a reagent or a chip for detecting the expression level of Sirt1 protein.

[0008] Furthermore, the kit is a reagent for detecting Sirt1 gene or protein expression level by any one of real-time fluorescence quantitative PCR, reverse transcription-polymerase chain reaction, in situ hybridization technology, biochip detection method, immunoblotting, enzyme-linked immunosorbent assay, mass spectrometry and colloidal gold immunoassay technology.

[0009] Furthermore, the kit includes an instrument or reagent for processing a sample, and the sample is selected from osteoblasts.

[0010] The present invention provides an application of a Sirt1 expression promoter in preparing a medicine for treating hormone-induced femoral head necrosis.

[0011] Furthermore, the Sirt1 expression promoter includes any one of a recombinant plasmid expressing or encoding a gene for Sirt1 protein, a recombinant virus expressing or encoding a gene for Sirt1 protein, a small molecule drug that increases the expression level of Sirt1 encoding mRNA or the expression level of Sirt1 encoding protein, an expression plasmid containing circRNA that regulates the expression level of Sirt1 encoding mRNA, a recombinant virus containing circRNA that regulates the expression level of Sirt1 encoding mRNA, and a miRNA inhibitor targeting the Sirt1 encoding mRNA sequence.

[0012] Furthermore, in the Sirt1 expression promoter, the recombinant plasmid expressing or encoding the gene of Sirt1 protein is an overexpression plasmid OE-Sirt1, and the sequence of the overexpression plasmid OE-Sirt1 is as follows (SEQ ID NO.1):

[0013]

[0014] Furthermore, the drug includes pharmaceutically acceptable excipients or carriers.

[0015] Furthermore, the auxiliary materials include one or more of an antioxidant, a pH regulator, a bactericide, an isotonic agent, a metal ion chelating agent, a buffer and a surfactant.

[0016] Furthermore, the carrier includes one or more of a diluent, a filler, a binder, an adsorption carrier and a disintegrant.

[0017] Furthermore, the steroid-induced femoral head necrosis is induced by dexamethasone.

[0018] In summary, compared with the prior art, the present invention has the following advantages and effects:

[0019] (1) The present invention provides the use of Sirt1 in the preparation of products for diagnosis, prognosis or treatment of hormonal osteonecrosis of the femoral head. Traditionally, the diagnosis of hormonal osteonecrosis of the femoral head mostly relies on imaging examinations (such as X-rays, CT, MRI) combined with the patient's clinical symptoms and medication history. These methods are difficult to achieve early warning and diagnosis of the disease. The present invention uses Sirt1 as a diagnostic marker, which is an innovation based on the molecular level. It can detect abnormalities in the early stages of the disease or even in the asymptomatic stage, so as to intervene in advance and effectively prevent the disease from worsening. For confirmed patients, continuous monitoring of Sirt1 expression levels can also be used to evaluate the treatment effect, enriching the diagnostic methods for hormonal osteonecrosis of the femoral head.

[0020] (2) The present invention provides an application of a Sirt1 expression promoter in the preparation of a drug for treating steroid-induced femoral head necrosis. Experiments have verified that overexpression of Sirt1 can significantly reduce the expression levels of ferroptosis regulatory proteins SLC7A11 and GPX4 in osteoblasts, thereby reducing the risk of steroid-induced avascular necrosis of the femoral head. This provides a potential therapeutic target for therapeutic products for steroid-induced femoral head necrosis, and has broad application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Figure 2 shows the ferroptosis in normal and dexamethasone-treated osteoblasts; A is the totaliron level in osteoblasts, B is the MDA level in osteoblasts, C is the mRNA expression of GPX4 in osteoblasts, D is the mRNA expression of SLC7A11 in osteoblasts, E is the fluorescence intensity of SLC7A11 in osteoblasts, and F is the fluorescence intensity of GPX4 in osteoblasts; Control represents the blank control group, Dex represents the dexamethasone-treated group, and * represents P < 0.05.

[0022] Figure 2Figure 2 is the expression of Sirt1 in normal and dexamethasone-treated osteoblasts; A is the mRNA expression of Sirt1 in osteoblasts, B is the protein expression of Sirt1 in osteoblasts, C is the distribution and fluorescence intensity of Sirt1 in osteoblasts detected by laser confocal microscopy, and D is the statistical graph of the fluorescence intensity of Sirt1 in osteoblasts detected by laser confocal microscopy; Control represents the blank control group, Dex represents the dexamethasone-treated group, and * represents P < 0.05.

[0023] Figure 3 is the expression of ferroptosis-related proteins in osteoblasts after overexpression of Sirt1; A is the mRNA expression of Sirt1 in osteoblasts, B is the protein expression of Sirt1 in osteoblasts, C is the protein expression of SLC7A11 in osteoblasts after overexpression of Sirt1, and D is the protein expression of GPX4 in osteoblasts after overexpression of Sirt1; Control represents the blank control group, OE-NC represents the overexpression negative control group, and OE-Sirt1 represents the Sirt1 gene overexpression group. Compared with the OE-NC group, *** P<0.001; compared with Dex+OE-NC group, # P<0.05.

[0024] Figure 4 Figure 2 shows the ferroptosis of osteoblasts after the use of Sirt1 inhibitor (Selisistat, EX527); A is the level of ROS in osteoblasts detected by DCFH-DA staining, B is the level of total iron in osteoblasts detected by spectroscopic breadth method, C is the level of MDA in osteoblasts detected by spectroscopic breadth method, D is the protein expression of Sirt1 in osteoblasts, E is the protein expression of SLC7A11 in osteoblasts after Sirt1 inhibition, and F is the protein expression of GPX4 in osteoblasts after Sirt1 inhibition; compared with the Control group, *P<0.05; compared with the Dex group, # P<0.05.

[0025] Figure 5is the role of Sirt1 hypermethylation in osteoblasts; A is the bioinformatics analysis of the methylation sites in the Sirt1 promoter region, B is the electrophoresis diagram of the DNA methylation level of the Sirt1 promoter in osteoblasts detected by nMS-PCR, C is the specific statistical diagram of the DNA methylation level of the Sirt1 promoter in osteoblasts, D is the mRNA expression of Sirt1 in osteoblasts, and E is the protein expression of Sirt1 in osteoblasts; Control represents the blank control group, Dex represents the dexamethasone-treated group, Dex+5-AzaC represents the dexamethasone and DNA methylation inhibitor (5-Azacytidine, 5-AzaC) group, compared with the Control group, *P<0.05; compared with the Dex group, # P<0.05. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0027] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0028] Example 1: Application of Sirt1 in the preparation of products for diagnosis, prognosis or treatment of hormone-induced femoral head necrosis.

[0029] 1. Experimental Materials and Methods

[0030] 1. Materials and reagents

[0031] Penicillin-streptomycin (Solarbio, China); gentamicin (Biotopped, Beijing); fetal bovine serum (Zhongqiao Xinzhou, China); α-EME culture medium (Zhongqiao Xinzhou, China); ROS kit (Servicebio, China); total RNA extraction kit (Axygen, USA); cDNA (complementary DNA) kit, RT-qPCR (real-time fluorescence quantitative PCR) kit (Takara, Japan); LipofectamineTM 2000 (Thermo Fisher Scientific, USA); goat anti-mouse / rabbit secondary antibodies (Zhongshan Jinqiao, China); Sirt1 overexpression plasmid was constructed by Shanghai Jima Biotechnology Co., Ltd.

[0032] 2. Instruments and equipment

[0033] Micropipette (Eppendorf, Germany); handheld vortex shaker (Kylin-bell, China); precision electronic balance (Sartorius, Germany); dual-temperature water bath shaker (HLD, Jiangsu); UV clean bench (Antai, China); desktop centrifuge (Eppendorf, Germany); biological safety cabinet (Sujing Antai, China); Biotek microplate reader (Dequan Xingye, Beijing); 6-well plate and 96-well plate (Seville, China); horizontal shaker (Kylin-bell, China); gradient PCR instrument (Eppendorf, Germany); fluorescence quantitative PCR instrument (Thermo Fisher Scientific, USA); gel imager (BIO-RAD, USA).

[0034] 3. Osteoblast Culture

[0035] 3.1 Cell recovery and culture

[0036] Prepare 37℃ warm water in a glass, take the mouse osteoblast (MC3T3-E1) cell line out of the -80℃ refrigerator, and immediately put it into warm water. The temperature of the warm water must be able to ensure that the cells in the cryopreservation tube melt quickly and present a complete cell suspension state. Prepare fresh α-EME medium (containing 10% fetal bovine serum, 1% double antibody), mix the cell suspension with the above medium and transfer it to a 15mL centrifuge tube, 1000rpm / min, centrifuge at room temperature for 5min to remove the cryopreservation solution. Add the prepared medium to the tube, gently re-suspend the cells with a pipette, and inoculate them in a 25cm 2 Place the cells in a culture bottle at 37°C, 95% O2, 5% CO2 incubator and culture for 24 hours. Observe under a microscope after 24 hours and replace the culture medium in time according to the cell adhesion and growth conditions.

[0037] 3.2 Cell passaging

[0038] Under the microscope, when the degree of confluence of cardiomyocytes reaches about 85%, they can be passaged. Aspirate the original culture medium, wash twice with pure culture medium, and then add 1mL of trypsin to the culture bottle to digest the cells. Under the microscope, after the cells are properly digested, aspirate the trypsin to terminate the digestion, and then add the prepared α-EME culture medium (containing 10% fetal bovine serum and 1% double antibody), blow and mix the cells, and passage the cells at the appropriate ratio.

[0039] 3.3 Cell cryopreservation

[0040] When the cells grow to 80% and are in the logarithmic growth phase, freeze them, discard the culture medium, add PBS to gently wash the cells twice, add 1mL of trypsin to digest, add the prepared α-EME culture medium to blow the cells again, and transfer them to a centrifuge tube, centrifuge at 1000rpm / min at room temperature for 5min, discard the supernatant in the tube and leave the required cell pellet, add an appropriate amount of pre-cooled cell freezing solution to the centrifuge tube, and gently mix the cells to make a cell mixture. The cell mixture in the centrifuge tube is divided into labeled cryopreservation tubes, 1.5mL per tube. Place the cryopreservation tube at 4℃ for 30 minutes, -20℃ for 2 hours, -80℃ overnight, and then transfer to liquid nitrogen for long-term storage.

[0041] 4. Real-time quantitative PCR (qRT-PCR)

[0042] 4.1 RNA extraction

[0043] ①Take out and collect the cultured osteoblasts, discard the culture medium in the bottle as much as possible, add 300μL of Buffer R-Ⅰ to each bottle, and repeatedly pipette at least 10 times;

[0044] ② Transfer the above cell suspension to a 1.5 mL centrifuge tube and aspirate repeatedly 10 times with a syringe equipped with a 25-gauge needle;

[0045] ③ Add 110 μL of Buffer R-Ⅱ, vortex for 30 seconds, and then centrifuge at 12000g, 4℃ for 5 minutes.

[0046] ④ After centrifugation, carefully aspirate the supernatant and place it in a 1.5 mL centrifuge tube. Add 200 μL of isopropanol and mix evenly with a pipette.

[0047] ⑤ Transfer the mixed solution in step ④ to the preparation tube, and place the preparation tube in a 2 mL centrifuge tube, centrifuge at 6000g, 4°C, for 1 min;

[0048] ⑥Discard the filtrate in the 2mL centrifuge tube, put the preparation tube back into the centrifuge tube, add 500μL Buffer W1A (with the specified volume of anhydrous ethanol added) into the preparation tube, and centrifuge at 12000g, 4℃ for 1min;

[0049] ⑦ Discard the filtrate again, then put the preparation tube back, add 700 μL of Buffer W2 (with the specified volume of anhydrous ethanol added), centrifuge at 12000g, 4℃ for 1 min; repeat step ⑦

[0050] ⑧Discard the filtrate, put the preparation tube back into a 2 mL centrifuge tube, and centrifuge at 12000g, 4°C for 1 min;

[0051] ⑨ Prepare a clean new 1.5mL centrifuge tube, put the preparation tube in, carefully add 100μL Buffer TE to the center of the preparation tube membrane, let it stand at room temperature for 1min, centrifuge at 12000g, 4℃ for 1min, and elute the RNA.

[0052] ⑩Ultraviolet spectrophotometer was used to measure the RNA concentration and A 260 / A 280 Ratio, stored at -80℃.

[0053] 4.2 Reverse transcription

[0054] ① Half an hour in advance, place the reagents and consumables required for reverse transcription in the clean bench and irradiate with ultraviolet light for 30 minutes;

[0055] ②Prepare the reverse transcription reaction system on ice, as shown in Table 1;

[0056] Table 1 Reverse transcription system (20 μL)

[0057]

[0058]

[0059] ③ Use vortex oscillation to fully mix the prepared reverse transcription system, centrifuge to remove bubbles and then perform reverse transcription reaction. The reverse transcription conditions are shown in Table 2.

[0060] Table 2 Reverse transcription conditions

[0061] Reaction temperature Reaction time 37℃ 15min 85℃ 5s 4℃ ∞

[0062] 4.3qRT-PCR reaction

[0063] ① Primer sequence

[0064] The gene ID and gene sequence were searched on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), entered into the PrimerBank website (https: / / pga.mgh.harvard.edu / primerbank / ) to search for the corresponding primer sequence and synthesized by Shanghai Bioengineering Co., Ltd. The primer sequences are shown in Table 3.

[0065] Table 3 Primer sequences

[0066]

[0067]

[0068] ②Prepare the reaction system according to the table below:

[0069] Table 4 qRT-PCR reaction system (20 μL)

[0070]

[0071] ③Get on the machine

[0072] After preparing the reaction system, the above solution was vortexed to mix, gently centrifuged, checked to make sure there were no bubbles, and then placed in a PCR instrument for amplification reaction. The reaction conditions are shown in Table 5.

[0073] Table 5 qRT-PCR reaction conditions

[0074]

[0075] ④Data analysis

[0076] After the qRT-PCR reaction, the relative expression of the target gene was calculated according to the melting curve and amplification curve analysis: -ΔΔCt Analyze the target gene content, where ΔΔCt = (Ct 目的基因 - Experimental group Ct 内参基因 )-(Control group Ct 目的基因 -Control group Ct 内参基因 ).

[0077] 5. Western blot

[0078] 5.1 Extraction of total cell protein

[0079] Prepare cell lysis buffer (Lysis Buffer: phosphatase inhibitor: protease inhibitor: 100mM PMSF=1000:10:1:5) according to the instructions in advance, mix well and place on ice for later use; remove cells, discard culture medium, add pre-cooled PBS to wash twice, shake several times during washing and then discard PBS in the bottle; add 1mL PBS to the culture bottle, scrape the cardiomyocytes with a cell scraper, move the cells and PBS to a centrifuge tube, centrifuge at 800g, 4℃ for 10min, resuspend the cells with pre-cooled PBS, repeat the operation twice at 800g, centrifuge at 4℃ for 5min; transfer the washed cells to a new pre-cooled centrifuge tube, add the above-prepared cold Lysis Buffer; shake vigorously on a shaker at 4℃ for 30s, place on ice for 4min, repeat five times; centrifuge at 12000g, 4℃ for 5min, and the supernatant after centrifugation is the whole protein extract. BCA method was used for quantification. The sample loading buffer was diluted with protein solution in a ratio of 1:5, mixed well, and denatured in a metal bath at 99°C for 5 min.

[0080] 5.2 Preparation of SDS-PAGE gel

[0081] Use the gel clamp to fix the thin and thick plates required for gel preparation on the gel strips, determine the required gel concentration according to the molecular weight of the target protein, and add the prepared separation gel and concentrated gel.

[0082] 5.3 Electrophoresis

[0083] Clamp the prepared gel plate with an electrophoresis stand, place it in the electrophoresis tank, and pour in the electrophoresis solution; add protein markers and samples to be tested to the corresponding wells by grouping using a micropipette; perform electrophoresis at a voltage of 80V throughout the process; end the electrophoresis when the protein position is close to the bottom.

[0084] 5.4 Electroporation

[0085] Cut the PVDF membrane to a size similar to the gel in advance and mark it, then place it in anhydrous ethanol to activate it; carefully use a spatula to cut the required part and soak it in the electrotransfer solution; then, clamp the electrotransfer clamp in the order of transfer cotton → filter paper → PVDF membrane → gel → filter paper → transfer cotton, place it in the electrotransfer tank, and add electrotransfer solution; set the electrotransfer conditions according to different protein molecular weights, turn on the power and start electrotransfer.

[0086] 5.5 Closure

[0087] After the transfer is completed, the PVDF membrane is placed in the prepared skim milk powder blocking solution (concentration is 5%); and the membrane is blocked on a shaker for 2 hours at room temperature.

[0088] 5.6 Antibody incubation

[0089] After blocking for 2 hours, remove the PVDF membrane and place it in the prepared PBST buffer; wash it 3 times on a horizontal shaker, 10 minutes each time; after washing, place it in the corresponding antibody (prepare the antibody of the target protein according to the instructions); incubate it on an inverted shaker at 4°C overnight; wash it 3 times with PBST buffer; after washing, place it in the goat anti-rabbit / mouse secondary antibody corresponding to the antibody; incubate it at room temperature for 2 hours.

[0090] 5.7 Exposure

[0091] After incubation with the secondary antibody for 2 hours, the membrane was removed and washed (washed 3 times with PBST buffer, 10 minutes each time); ECL colorimetric solution A and solution B were prepared into a developer in a ratio of (1:1); under light-proof conditions, the membrane was placed in the developer and dipped evenly; the membrane was removed and placed on the film carrier plate of the gel imager, and the instrument was started for exposure; subsequently, the Image Lab software was used to analyze and count the bands.

[0092] 6. Immunofluorescence

[0093] ① After each group of cells received the corresponding treatment according to the planned experimental conditions, the culture medium was removed, and the cells were rinsed twice with PBS, each time for 5 minutes, and the cells were fixed with 4% paraformaldehyde at room temperature for 10 minutes, and the cell membrane was permeabilized with 0.5% Triton X-100 for 20 minutes.

[0094] ② Wash with PBS three times, 5 min each time, and block with 5% BSA at room temperature for 1 h.

[0095] ③ Wash with PBS three times, 5 min each time, add primary antibody prepared in PBS, and incubate at 4°C overnight.

[0096] ④ Wash with PBS three times, 5 min each time, add corresponding fluorescent secondary antibody, and incubate at room temperature for 2 h in the dark.

[0097] ⑤ Wash with PBS three times, 5 min each time, add DAPI solution, and incubate at room temperature for 15 min in the dark.

[0098] ⑥ Wash with PBS 3 times, 5 min each time. Observe and photograph the samples under a fluorescence microscope, and use the SNT plug-in of Image J to track and analyze the cells positive for immunofluorescence staining.

[0099] 7. Intracellular ROS Determination

[0100] Intracellular ROS was measured using 2,7′-dichlorodihydrofluorescein (DCFH-DA, Servicebio). After treatment, cells were stained with DCFH-DA for 30 min, washed three times with PBS, and stained with DAPI for 10 min before the fluorescence of cells in PBS was detected using a confocal laser scanning microscope.

[0101] 8. Determination of total cellular iron

[0102] Total iron levels were determined using an iron colorimetric assay kit purchased from NJJCBIO. According to the manufacturer's instructions, cells were added to the iron colorimetric reagent, soaked in boiling water for 5 min, and centrifuged at 3500 g for 10 min at 4 °C to obtain the supernatant or assay. 200 μL of the sample was incubated with a 96-well microplate and the absorbance was measured at 520 nm using a microplate reader.

[0103] 9. Determination of cell malondialdehyde (MDA)

[0104] Discard the cell culture supernatant, scrape the cells with a cell scraper, and transfer the cells to a plastic centrifuge tube with a pipette. Add 0.5 mL of the extract, mix for 2 minutes, break the cells into a suspension, sample 0.1 mL in a 1.5 mL centrifuge tube, and add 1000 μL of the working solution. Mix with a vortex mixer, bathe at 95°C for 40 minutes, take out and cool, centrifuge at 4000 rpm for 10 minutes, accurately pipette 0.25 ml of the reaction solution from each tube into a new 96-well plate, and measure the absorbance of each well with an ELISA reader (530 nm).

[0105] 10. Nested methylation specific PCR (nMS-PCR)

[0106] The whole genome DNA of normal osteoblasts and dexamethasone-treated osteoblasts was extracted according to the instructions of the DNA extraction kit, and the whole genome DNA was methylated. The changes in the degree of DNA methylation in the promoter region of the Sirt1 gene were detected by nMS-PCR. For the Sirt1 gene sequence, 1 external primer and 2 internal primers were designed online (https: / / www.urogene.org / cgi-bin / methprimer / methprimer.cgi), and the primer sequences are shown in Table 6.

[0107] Table 6 Sirt1 nMS-PCR primers

[0108]

[0109]

[0110] 11. Statistical processing

[0111] The results were analyzed and processed using Prism 9.0 statistical software and expressed as mean ± standard deviation (Mean ± SD). The Student's t test was used to compare the means of two samples, the One-way ANOVA test was used to compare the means of multiple samples, and the Student-Newan-Keuls test was used to compare the means between groups. P ≤ 0.05 was considered statistically significant.

[0112] 2. Experimental Results

[0113] 1. Use corresponding biochemical kits, real-time fluorescence quantitative PCR and immunofluorescence to detect changes in the levels of iron death-related indicators in osteoblast samples under the action of dexamethasone

[0114] The corresponding biochemical kits were used to detect the levels of total iron and iron death-related marker MDA in osteoblasts. The results showed that compared with the Control group, the levels of total iron and MDA in the dexamethasone group were significantly increased ( Figure 1 A, B); Real-time fluorescence quantitative PCR and immunofluorescence were used to detect the expression of osteoblast ferroptosis-related genes SLC7A11 and GPX4. The results showed that compared with the Control group, the protein expression of ferroptosis-related genes GPX4 and SLC7A11 in the dexamethasone group was significantly decreased ( Figure 1 CF). This indicates that dexamethasone can promote osteoblast ferroptosis ( Figure 1 ).

[0115] 2. Real-time fluorescence quantitative PCR, protein immunoblotting and laser confocal microscopy were used to detect the expression of Sirt1 in osteoblasts under the action of dexamethasone

[0116] Real-time fluorescence quantitative PCR and protein immunoblotting were used to detect the expression of Sirt1 mRNA and protein in osteoblasts under the action of dexamethasone. The results showed that the expression of Sirt1 in osteoblasts exposed to Dexamethasone was decreased ( Figure 2 A, B); Laser confocal microscopy showed that Sirt1 was mainly distributed in the cell nucleus, and the fluorescence signal intensity of Dex on osteoblasts was weaker than that in the control group ( Figure 2 C, D), indicating that Dex inhibited the expression of Sirt1 ( Figure 2 ).

[0117] 3. Real-time fluorescence quantitative PCR and protein immunoblotting were used to detect changes in the levels of ferroptosis-related indicators in osteoblast samples after overexpression of Sirt1

[0118] First, the Sirt1 overexpression plasmid (OE-Sirt1, constructed by Shanghai Genetech) was used to infect osteoblasts, and its transfection efficiency was verified by real-time fluorescence quantitative PCR and protein immunoblotting. Figure 3 A, B), and found that the mRNA and protein levels of osteoblasts overexpressing Sirt1 were significantly upregulated. Western blotting was used to detect the protein expression levels of SLC7A11 and GPX4 after overexpression of Sirt1, and it was found that upregulation of Sirt1 enhanced the expression of SLC7A11 and GPX4 in Dex-induced osteoblasts ( Figure 3 C, D). These results suggest that overexpression of Sirt1 can inhibit Dex-induced ferroptosis of osteoblasts ( Figure 3 ).

[0119] 4. Use confocal laser scanning microscopy, biochemical kits, and protein immunoblotting to detect changes in the levels of ferroptosis-related indicators in osteoblast samples after silencing Sirt1

[0120] To further verify the role of Sirt1 in Dex-induced osteoblast ferroptosis, osteoblasts were treated with Sirt1 inhibitor (EX-527). The study found that the ROS content, total iron and MDA levels in the Dex+EX-527 group were higher than those in the Dex group ( Figure 4 AC), Western blotting detected that silencing Sirt1 reduced the protein expression of Sirt1, SLC7A11 and GPX4 in osteoblasts induced by Dex ( Figure 4 DF), the above results indicate that silencing Sirt1 promotes Dex-induced ferroptosis of osteoblasts ( Figure 4 ).

[0121] 5. Effect of Sirt1 hypermethylation on Sirt1 expression in osteoblasts

[0122] DNA methylation is a highly conservative epigenetic modification, which refers to the covalent binding of methyl groups to the 5'-CpG-3' dinucleotide (5-methylcytosine) of DNA, thereby affecting the stability of the genome, gene expression and development. In view of the key role of DNA methylation in gene transcription, the inventors first used bioinformatics software to analyze and predict the structure of the Sirt1 promoter region and found that there were two CpG islands ( Figure 5 A). Subsequently, nested methylation-specific PCR (nMS-PCR) was used to detect changes in DNA methylation in the Sirt1 promoter region, and it was found that compared with the Control group, the Sirt1 DNA methylation level in the Dex group was increased, and 5-AzaC (5-Azacytidine, a DNA methylation inhibitor) further inhibited this process ( Figure 5 B, C). In order to further understand the effect of DNA methylation on Sirt1 expression, the present inventors used 5-AzaC to treat osteoblasts and found that compared with the Dex group, the Sirt1 mRNA and protein levels in the 5-AzaC group were increased ( Figure 5 D, E), indicating that Dex upregulated the Sirt1 DNA methylation level and downregulated the Sirt1 expression in osteoblasts ( Figure 5 ).

[0123] In summary, the present invention found that dexamethasone promotes osteoblast ferroptosis. Under dexamethasone treatment, Sirt1 transcription and protein expression levels in osteoblasts are downregulated, and Sirt1 DNA methylation levels are upregulated. Sirt1 is one of the potential targets of dexamethasone in promoting hormonal osteonecrosis of the femoral head. Overexpression of Sirt1 can inhibit the effect of dexamethasone-induced osteoblast ferroptosis. At the same time, silencing Sirt1 using a Sirt1 inhibitor (EX-527) can promote Dex-induced osteoblast ferroptosis. For the first time, it is confirmed that Sirt1 has a good diagnostic value for hormonal osteonecrosis of the femoral head, and can be used as a potential biomarker for the diagnosis and prognosis of hormonal osteonecrosis of the femoral head. It enriches the theoretical basis for the diagnosis of hormonal osteonecrosis of the femoral head using molecular biological methods. At the same time, it is proposed for the first time that Sirt1 expression promoters are used in the preparation of drugs for the treatment of hormonal osteonecrosis of the femoral head, which is of great significance.

Claims

1. Application of Sirt1 in the preparation of products for diagnosis, prognosis or treatment of hormone-induced femoral head necrosis.

2. The use according to claim 1, characterized in that Sirt1 is used to prepare a chip, a kit or a nucleic acid membrane strip for diagnosing or prognosing hormone-induced femoral head necrosis.

3. The use according to claim 1, characterized in that The application of the Sirt1 expression promoter in the preparation of a drug for treating hormone-induced femoral head necrosis.

4. The use according to claim 3, characterized in that The Sirt1 expression promoter includes any one of a recombinant plasmid expressing or encoding a gene for Sirt1 protein, a recombinant virus expressing or encoding a gene for Sirt1 protein, a small molecule drug that increases the expression level of Sirt1 encoding mRNA or the expression level of Sirt1 encoding protein, an expression plasmid containing circRNA that regulates the expression level of Sirt1 encoding mRNA, a recombinant virus containing circRNA that regulates the expression level of Sirt1 encoding mRNA, and a miRNA inhibitor targeting the Sirt1 encoding mRNA sequence.

5. The reagent according to claim 4, characterized in that The recombinant plasmid expressing or encoding the gene of Sirt1 protein is the overexpression plasmid OE-Sirt1, and the sequence of the overexpression plasmid OE-Sirt1 is shown in SEQ ID NO.

1.

6. The use according to claim 3, characterized in that The drug includes pharmaceutically acceptable excipients or carriers.

7. The use according to claim 3, characterized in that The auxiliary materials of the medicine include one or more of antioxidants, pH regulators, bactericides, isotonic agents, metal ion chelating agents, buffers and surfactants.

8. The use according to claim 3, characterized in that The carrier of the drug includes one or more of a diluent, a filler, a binder, an adsorption carrier and a disintegrant.

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