Application of Fbxl16 protein and gene recombinant vector in preparing anti-inflammatory drugs

Abstract

The present invention belongs to the technical field of protein drugs, and discloses the application of Fbxl16 protein and gene recombinant vectors in the preparation of anti-inflammatory drugs. The present invention points out that the Fbxl16 protein can effectively improve the inflammatory condition and play an anti-inflammatory effect, and can be used as an anti-inflammatory drug in the treatment of inflammatory diseases such as rheumatoid arthritis. At the same time, by constructing a gene recombinant vector containing the Fbxl16 gene, the corresponding gene drug can be developed. In addition, the present invention also proposes that licorice chalcone B (LCB) can act as an Fbxl16-targeted activator to enhance the anti-inflammatory effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein drugs, and in particular relates to the application of Fbxl16 protein and a gene recombination vector in the preparation of anti-inflammatory drugs. Background Art

[0002] RA (rheumatoid arthritis) is one of the most common chronic inflammatory diseases, characterized by autoimmunity, severe synovial and systemic inflammation, cell proliferation, soft tissue swelling, stiffness, and bone erosion in the joints. These inflammatory processes can ultimately lead to severe disability and premature death. Despite recent advances in the clinical treatment of RA, the toxicity of drug therapy remains a significant challenge. In current clinical practice, treatment methods or treatment combinations used vary depending on the type of arthritis. The most widely used drugs, such as the disease-modifying antirheumatic drugs MTX and hydroxychloroquine (Plaquenil), can slow or prevent the immune system's attack on the body's own joint tissues. However, long-term systemic medications often lead to drug resistance and off-target effects. In addition, physical therapy can also help relieve arthritis pain, but once conservative measures are no longer effective, patients may require surgical treatment such as joint repair, fusion, or even replacement.

[0003] Therefore, the development of more anti-inflammatory drugs with high efficacy and specific targeting characteristics is crucial for the treatment of inflammatory diseases such as RA (rheumatoid arthritis). Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes the use of Fbxl16 protein and a genetically recombinant vector in the preparation of anti-inflammatory drugs. The present invention points out that Fbxl16 protein can effectively improve inflammatory conditions and exert anti-inflammatory effects. It can be used as an anti-inflammatory drug in the treatment of inflammatory diseases such as rheumatoid arthritis. At the same time, by constructing a genetically recombinant vector containing the Fbxl16 gene, corresponding gene drugs can be developed. In addition, the present invention also proposes that licorice chalcone B (LCB) can be used as a targeted activator of Fbxl16 to enhance the anti-inflammatory effect.

[0005] The present invention provides the use of Fbxl16 protein in the preparation of anti-inflammatory drugs.

[0006] The present invention reports that Fbxl16 (F-box and leucine rich repeat protein 16) mRNA levels are significantly downregulated in the blood of patients with rheumatoid arthritis (RA) and rats with adjuvant-induced arthritis (AIA), compared to healthy individuals. Further experiments have shown that Fbxl16 can inhibit the production of proinflammatory cytokines and promote the expression of anti-inflammatory factors, exerting anti-inflammatory effects. Therefore, Fbxl16 protein has significant potential for the development of anti-inflammatory drugs.

[0007] The present invention also provides the use of Fbxl16 protein in preparing medicine for treating arthritis.

[0008] Preferably, the arthritis is rheumatoid arthritis.

[0009] Fbxl16 protein can reduce the degree of foot swelling and arthritis score in adjuvant-induced arthritis rat model (AIA rats), alleviate bone destruction, and inhibit synovial hyperplasia, and therefore can be used to prepare drugs for the treatment of rheumatoid arthritis.

[0010] The present invention also provides the use of a recombinant gene vector containing the Fbxl16 gene in the preparation of an anti-inflammatory drug. The recombinant gene vector is constructed from an expression vector and the Fbxl16 gene. The Fbxl16 gene sequence is referenced by NCBI sequence number NM_153350. The recombinant gene vector can exert an anti-inflammatory effect through overexpression of Fbxl16 and has outstanding potential as a gene drug.

[0011] Preferably, the expression vector is plasmid DNA or adeno-associated virus.

[0012] The present invention also provides the use of a gene recombination vector containing the Fbxl16 gene in preparing a drug for treating arthritis.

[0013] The present invention also provides use of the Fbxl16 targeted activator in preparing a drug for treating arthritis.

[0014] Preferably, the Fbxl16 targeted activator is licochalcone B.

[0015] Licorice chalcone B (BLCB), a chalcone found in licorice root, is generally believed to have therapeutic effects in cardioprotection, Alzheimer's disease (AD), antioxidant and free radical protection, and inducing cancer cell apoptosis. The present invention indicates that licorice chalcone B (LCB) can activate and increase Fbxl16 transcription, upregulating gene and protein levels, and therefore can be used as a targeted activator of Fbxl16. Furthermore, licorice chalcone B can inhibit cell invasion and promote apoptosis, improve inflammatory markers in the synovium, and reduce IL-17 / CD4 expression in the spleen, demonstrating immunomodulatory and anti-inflammatory properties.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention proposes that the gene level of Fbxl16 is significantly downregulated in both RA patients and adjuvant-induced arthritis rats, and its overexpression can significantly inhibit the level of proinflammatory factors secreted by synovial cells induced by LPS, indicating that the Fbxl16 protein has an anti-inflammatory effect. The present invention also provides evidence that injecting a recombinant gene vector containing the Fbxl16 gene into the knee joint can significantly inhibit the foot swelling volume, arthritis score and proinflammatory cytokine production in adjuvant-induced arthritis rats. In addition, the present invention also successfully discovered that licorice chalcone B (LCB) can be used as a targeted activator of Fbxl16, which activates and increases the transcription of Fbxl16, upregulating gene expression and protein levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The normalized expression scatter plots of each gene in the blood of RA patients (A) and AIA rats (B) are shown.

[0019] Figure 2 Figure 3. Fbxl16 mRNA (A) and protein levels (B) in RAFLS cells after TNF-α treatment, Fbxl16 transcriptional activity under stimulation with LPS, Pam3, poly(I:C), IL-1β, and TNF-α, and transcriptional activity in the presence of TNF-α when Lef, Chlo, Tet, or Nab were added (C).

[0020] Figure 3 Figure 2 shows the expression of inflammatory factors in MH7A cells overexpressing Fbxl16 in the presence of TNF-α.

[0021] Figure 4 Arthritis score (A) and paw swelling volume (B) of AIA rat model after treatment with adenovirus Ad-Fbxl16.

[0022] Figure 5Micro-CT radiological images (A) and BV, BS, TMD, TMD and Micro-CT scores (B) in the AIA rat model after treatment with adenovirus Ad-Fbxl16.

[0023] Figure 6 Figure 3 H&E staining of sagittal sections of the knee joints of rats in each experimental group (A), immunofluorescence images of synovial tissue (B), and serum cytokine levels (C).

[0024] Figure 7 Figure 3 Fbxl16 mRNA (A) and protein levels (B) in RAFLS cells after LCB treatment, and Fbxl16 transcriptional activity in HEK293 cells (C).

[0025] Figure 8 is the relative mRNA expression levels of IL-6 and IL-1β in RAFLS cells in the presence of LPS or TNF-α.

[0026] Figure 9 Figure 3 shows the invasion and apoptosis of RASFs cells after LCB treatment in the presence of LPS (A), and the expression levels of IL-1β and IL-6 after Fbxl16 knockout and LCB treatment (B).

[0027] Figure 10 Arthritis scores (A) and paw swelling volumes (B) in AIA rats after oral administration of LCB.

[0028] Figure 11 Erythrocyte sedimentation rate (A) and expression levels of IL-6 and IL-1β in synovial tissue of AIA rats after oral administration of LCB (B).

[0029] Figure 12 Micro-CT radiographic images of bone erosion and trabecular bone in rats treated with LCB (A), and immune-related indicators of spleen single cells (B). DETAILED DESCRIPTION

[0030] In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are only preferred embodiments of the present invention and do not limit the scope of protection claimed by the present invention. Any modification, substitution, or combination made without violating the spirit and principle of the present invention is included in the scope of protection of the present invention.

[0031] Unless otherwise specified, the raw materials, reagents, or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0032] Example 1: Anti-inflammatory effect of Fbxl16 protein

[0033] 1. Total RNA Extraction from Blood Samples

[0034] Blood samples were collected from patients with RA and healthy volunteers at the Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou (China), with written informed and voluntary consent and approval from the Research Ethics Committee (GDREC 2015391H). All epidemiological investigations and classifications from volunteers were conducted according to the criteria of the American College of Rheumatology. Wild-type male Sprague-Dawley (SD) rats, weighing 80–120 g, were purchased from the Laboratory Animal Services Center of the Chinese University of Hong Kong, China. Animal care and handling procedures were in accordance with institutional guidelines and animal regulations (Department of Health, Hong Kong Special Administrative Region, China).

[0035] Total RNA from blood was isolated using the FavorPrep™ Blood / Cultured Cell Total RNA mini kit (Favorgen Biotech). Add 200-300 μL of fresh, anticoagulant-preserved whole blood to a microcentrifuge tube. Mix 5 volumes of RL buffer with 1 volume of sample, invert to mix thoroughly, and incubate on ice for 10 minutes, briefly vortexing twice. Pellet the cells by centrifugation at 4500 rpm for 1 minute. Discard the supernatant, add 600 μL of RL buffer, briefly vortex to resuspend the cell pellet, and centrifuge at 4500 rpm for 1 minute to repel the cells again. Discard the supernatant. Add 350 μL of FARB buffer and 3.5 μL of β-mercaptoethanol to the cell pellet and vortex vigorously for 1 minute to completely resuspend the cells. Place the filter column in a collection tube, transfer the sample mixture to the filter column, and centrifuge at maximum speed for 2 minutes. Transfer the clarified supernatant from the collection tube to a new microcentrifuge tube, measure the volume of the supernatant, add 1 volume of 70% ethanol, and vortex to mix thoroughly. Place the FARB column in the collection tube, transfer the ethanol-added sample mixture (including any precipitate) to the FARB column, centrifuge at full speed for 1 minute, discard the flow-through, and replace the FARB column. Return the FARB column to the collection tube, add 500 μL of wash buffer 1 to the FARB column, centrifuge at full speed for 1 minute, discard the flow-through, and return the FARB column to the collection tube. Add 750 μL of wash buffer 2 to the FARB column, centrifuge at full speed for 1 minute, discard the flow-through, and return the FARB mini column to the collection tube. Repeat once. Centrifuge the FARB column at full speed for 3 minutes. Place the FARB column in the elution tube and add 40-100 μL of RNase-free ddH2O to the center of the FARB column membrane. RNA quality and concentration were measured using UV spectrophotometry (NanoDrop Technologies, USA). 1 μg of RNA was reverse transcribed using Maxima H Minus cDNA Synthesis Master Mix (Thermo, USA). The mixture was incubated at 25°C for 10 minutes, then at 50°C for 15 minutes, and the reaction was terminated by heating at 85°C for 5 minutes. A 20 μL PCR mixture consisted of 0.5 μL template, 0.5 μL F primer (5'-CTGCCATCTCACAACTTCTA-3' (SEQ ID NO: 1)) and R primer (5'-ACATACTCCAGTGCCATATC-3' (SEQ ID NO: 2)), 0.5 μL template, 10 μL SYBR Master Mix, and 8.5 μL ddH2O, for a total of 20 μL.Gene expression was quantified using the ViiA 7 Real-Time PCR System (Applied Biosystems). All mRNA data were normalized to the reference gene β-actin and relative quantification was performed using the ΔΔCT method.

[0036] Figure 1 For RA patients (such as Figure 1 (A)) and AIA rats (as shown in Figure 1 (B) Normalized expression scatter plot of each gene in blood, the center line represents unchanged gene expression, the dashed line represents the 3-fold regulation threshold, and the data points outside the dashed line and the data points in the lower right part meet the selected regulation threshold. Figure 1 The results showed that compared with the healthy control group, the mRNA level of Fbxl16 was significantly downregulated in the blood of RA patients and AIA (adjuvant-induced arthritis) rats.

[0037] TNF-ɑ induces a decrease in Fbxl16 gene, protein, and transcript levels

[0038] RASFs (rheumatoid arthritis synovial fibroblasts) were seeded in 6-well plates and stimulated with 10 or 20 ng / mL TNF-α for 24 hours the following day. Gene and protein expression was determined using Fbxl16-specific primers (F primer: 5'-CTGCCATCTCACAACTTCTA-3' (SEQ ID NO: 1); R primer: 5'-ACATACTCCAGTGCCATATC-3' (SEQ ID NO: 2)) and an antibody (FBXL16 Polyclonal Antibody (ThermoFisher, PA5-21094)). Samples were lysed on ice for 15 minutes using protein extraction buffer (ab156034). After centrifugation at 13,000 g for 15 minutes at 4°C, the supernatant was collected and quantified using Protein Assay Dye Reagent (Bio-Rad). Protein concentrations were measured spectrophotometrically at a wavelength of 595 μm. A 20 μg protein sample was analyzed by 12% SDS-PAGE and then transferred to a PVDF membrane (Bio-Rad). The membrane was blocked with 5% BSA for 1 hour and incubated overnight at 4°C with anti-Fbxl16 antibody (ThermoFisher; antibody: PBST = 1:1000) and anti-GAPDH antibody (Santa Cruz Biotechnology, antibody: PBST = 1:2000). After washing with TBST, the membrane was incubated with anti-rabbit and anti-mouse secondary antibodies (Santa Cruz Biotechnology, antibody: PBST = 1:2000) at room temperature for 2 hours. An ultrasensitive ECL chemiluminescence reagent kit (Beijing 4A Biotec h Co., Ltd.) was used to detect bands on an Amersham Imager 600 (GE) imaging system, and the grayscale of the target bands was analyzed using ImageJ software.

[0039] Construction of pEZX-PL01-Fbxl16 plasmid

[0040] Using the pHBAAV-CMV-MCS-3flag-T2A-ZsGreen plasmid as the gene vector, add each reagent in the order listed in Table 1, gently pipette to mix, and place in a 37°C water bath for 1-2 hours. After enzyme digestion, perform agarose gel electrophoresis to recover the linearized vector.

[0041] Table 1 Vector enzyme digestion system

[0042]

[0043]

[0044] PCR amplification of target gene fragments

[0045] The amplification system was prepared using the contents of Table 2, mixed gently, and placed in a PCR instrument for amplification using the PCR program shown in Table 3 to amplify the target gene fragment (Fbxl16 gene, its sequence refers to NCBI sequence number NM_153350).

[0046] Table 2 PCR amplification system

[0047]

[0048] Table 3 PCR program

[0049]

[0050] Target gene fragment is connected to linearized vector

[0051] Prepare the reaction system shown in Table 4 below in an ice-water bath, react at 50°C for 30 min, and place on ice for 5 min to obtain the ligation product, which is then immediately subjected to subsequent transformation.

[0052] Table 4 Reaction system

[0053]

[0054] conversion

[0055] 1) Take the DH5α competent cells out of the -80°C freezer and immediately thaw them on ice. Be gentle during the competent cell aliquoting process to minimize mechanical damage.

[0056] 2) After the competent medium has thawed, aliquot the solution into 50 μL aliquots. Add the ligation product to a volume no greater than 1 / 10 of the competent medium volume and place on ice for 20-30 minutes.

[0057] 3) Heat shock at 42°C for 90 seconds. Immediately place the tube on ice and incubate for 2-3 minutes.

[0058] In a clean bench, add 500 μL of LB medium and gently invert 3-5 times;

[0059] 5) Incubate at 37°C, 230 rpm, shaking for 45-60 min;

[0060] 6) Apply the bacterial solution evenly to a solid plate of the corresponding resistance, then incubate the plate upside down at 37°C in a constant temperature incubator for 12-16 hours to complete the construction of the pEZX-PL01-Fbxl16 plasmid.

[0061] Using liposomes ( The pEZX-PL01-Fbxl16 recombinant plasmid was transferred into HEK293 cells using a transfection reagent (Invitrogen, 3000) and seeded into 24-well plates. Each well contained 2 × 10 HEK293 cells. 5 Two treatments were performed: ① LPS, TNF-α, IL-1β, Pam3CSK4 (Pam3), and poly(I:C)HMW were added to each well and the cells were incubated at 37°C for 24 hours. ② After pretreatment with leflunomide (Lef), chlortetracycline (Chlo), tetracycline (Tet), and nalbuphine (Nab) for 2 hours, TNF-α was added and the cells were incubated for an additional 24 hours. The culture medium was removed, and the cells were rinsed in 1× PBS. All the rinse solution was removed, and 1× PLB was added to lyse the cells at room temperature for 15 minutes. 10μL of cell lysis buffer was added to 100μL of LAR II, and the mixture was mixed by pipetting. The reading was then taken, which was the Firefly luciferase value. 100μL of Stop&Glo was added and the reading was taken again, which was the Renilla luciferase value. Data processing: First, calculate the ratio of Firefly luciferase / Renilla luciferase in each tube. Then, taking the ratio of the control group as 1, the relative luciferase activity of the different treatment groups can be obtained, that is, the regulatory activity of gene transcription in the treatment group.

[0062] Depend on Figure 2 As shown in (A) and (B), under TNF-α stimulation, the mRNA and protein levels of Fbxl16 in RAFLS (rheumatoid arthritis fibroblast-like synoviocytes) cells were significantly reduced. Dual-luciferase reporter assays confirmed that the transcriptional activity of Fbxl16 in HEK293 cells was significantly inhibited in the presence of Toll-like receptor ligands such as lipopolysaccharide (LPS), Pam3CSK4 (Pam3), poly (I: C) HMW (poly (I: C)) and proinflammatory cytokines (TNF-α and IL-1β). In contrast, the expression of Fbxl16 in HEK293 cells was significantly inhibited by TNF-α. Figure 2 As shown in (C), in the presence of TNF-α, the transcriptional activity of Fbxl16 was enhanced by the addition of DMARDs or anti-inflammatory drugs, such as leflunomide (Lef), chlortetracycline (Chlo), tetracycline (Tet), and nalbupone (Nab).

[0063] 3. Real-time quantitative PCR detection of Fbxl16 overexpression to inhibit the expression of inflammatory factors

[0064] MH7A cells (2×10 6 cells / well) were seeded on 6-well plates and cultured overnight to allow them to adhere to the wall. The pEZX-PL01-Fbxl16 recombinant plasmid was transformed into MH7A cells using a transfection reagent (Invitrogen, 3000), incubated at 37°C for 24 hours, and then cultured with TNF-α in a 6-well plate for another 24 hours. Total RNA was extracted using the FavorPrep Total RNA purification mini kit (Favorgen, PingTung, Taiwan). VILOMaster Mix (Invitrogen, Scotland, UK) was used to synthesize cDNA by reverse transcription, and the gene expression levels of inflammatory factors IL-6, IL-1β, and TNF-α were detected by real-time quantitative PCR.

[0065] like Figure 3 As shown in the results, after overexpression of Fbxl16, attenuated mRNA levels of IL-6 and TNF-α were observed in TNF-α-induced MH7A cells, confirming the anti-inflammatory effect of Fbxl16 protein.

[0066] Example 2: Overexpression of Fbxl16 inhibits inflammatory response and bone destruction in AIA rats

[0067] 1. Achieve high expression of Fbxl16 mediated by adenovirus in AIA rat model

[0068] This study used male Sprague-Dawley rats, 4-6 weeks old, purchased from the Guangdong Medical Laboratory Animal Center and weighing 80-120 g. The animals were housed in a room equipped with a temperature-controlled, automated ventilation system and a 12-hour light / dark cycle, with free access to food and water. This study was approved by the Animal Ethics Committee of the Health Bureau of the Macao Special Administrative Region of China and conducted in accordance with the Guidelines for Animal Care and User Committees of the Macau University of Science and Technology. Complete Freund's adjuvant (100 μL) of mineral oil (Sigma, USA) containing 2.5 mg / mL of inactive Mycobacterium tuberculosis was emulsified after prolonged grinding, and the emulsified oil was injected intradermally into the base of the rat's tail. The first bout of inflammation occurred approximately nine days after adjuvant injection, and the rat's paw volume was measured and recorded every three days to establish an adjuvant-induced arthritis rat model (AIA rat).

[0069] Thirty-two male rats were randomly divided into five experimental groups: (1) healthy rat control group (n = 6), which received no treatment; (2) adjuvant-induced arthritis model control group (n = 6); (3) MTX (methotrexate) positive control group (n = 6), which received 7.6 mg / kg / week of MTX via gavage; (4) adenovirus Ad-GFP (green fluorescent protein) group (n = 6), which injected 100 μL of adenovirus Ad-GFP into the joint cavity of AIA rats using a microsyringe; and (5) adenovirus Ad-Fbxl16 group (n = 8), which injected 100 μL of adenovirus Ad-Fbxl16 into the joint cavity of AIA rats using a microsyringe. After the experimental groups were set up, the rats were observed daily to obtain arthritis scores and paw swelling volume information for each experimental group. At the end of the treatment course, the rats were sacrificed, and blood and some organs were collected. The right hind foot was photographed and frozen, and the left hind foot was amputated and fixed in 4% PFA.

[0070] The main construction methods of adenovirus Ad-GFP and adenovirus Ad-Fbxl16 are:

[0071] 1) Day 1: Subculture AAV-293 cells into 100 mm dishes for transfection. Place in an incubator at 37°C, 5% CO2, and 95% relative humidity.

[0072] 2) Day 3: Transfection can be performed when the cell density reaches about 80-90% confluence.

[0073] Lipofectamine transfection: Preheat OptiMEM in a 37°C water bath. Lipofectamine™ transfection reagent must be returned to room temperature before use and shake well before use. The transfection complex components are shown in Table 5.

[0074] Table 5 Transfection complex

[0075]

[0076] 3) Medium change: 6 h after transfection, replace with fresh complete medium containing 10% fetal bovine serum (FBS).

[0077] 4) Cell collection: 72 h after transfection, cells containing AAV particles were gently scraped off with a cell scraper and collected in a 15 mL centrifuge tube. The cells were centrifuged at 150 × g for 3 min, the culture supernatant was removed, and the tube was washed once with PBS. Finally, the cells were resuspended in 300 μL of PBS.

[0078] 5) Cell disruption: Prepare a 37°C water bath and liquid nitrogen. Repeat the freeze-thaw cycle three times in a 37°C water bath containing cells. Centrifuge at 2000 × g for 5 min at 4°C to remove cell debris and collect the supernatant containing AAV particles.

[0079] 6) Purification: Add 0.1 μL Benonase enzyme to each 1 mL of lysis supernatant and incubate in a 37°C water bath for 1 hour to remove the cell genome and residual plasmid DNA in the virus solution. Centrifuge at 600×g, 4°C for 10 minutes and take the supernatant. Perform column purification according to the Biomiga Adeno-Associated Virus Purification Kit V1469-01. Add 4 mL of AAV virus sample liquid obtained by column purification to an ultrafiltration tube and centrifuge at 1400×g for 30 minutes to obtain approximately 200 μL of AAV. Collect the final purified virus and store it at -80°C.

[0080] Depend on Figure 4 It can be seen that the anti-inflammatory effect of Fbxl16 protein was further verified by injecting adenovirus Ad-Fbxl16 into the knee joints of AIA rats. Although AIA rats injected with adenovirus Ad-GFP showed joint stiffness and severe foot swelling, those injected with Ad-Fbxl16 (1×10 11 PFU / mL) of AIA rats, the arthritis scores and paw swelling volume were significantly attenuated.

[0081] 2. MicroCT Analysis of Rat Foot Bone Destruction

[0082] The right hind foot was thawed on ice and scanned using a micro-CT scanner (SkyScan 1176, Bruker, Belgium). Scanning parameters were 35 μm resolution, 62 kV, 385 μA, 98 ms exposure time, 0.70 angular velocity, and an Al 1 mm filter. After scanning, images were reconstructed using NRecon software (Bruker-micro CT, Belgium). CTvox software was used to open the reconstructed data files and generate observable three-dimensional images. CTAn software was used to analyze the scan data.

[0083] The microCT score is derived from five disease-related parameters assessed by microcomputed tomography (microCT) scanning: bone mineral density, bone volume fraction, cortical mineral density, trabecular number, and total porosity. The formula for calculating the microCT score is as follows: (obtained value - minimum value) / (maximum value - minimum value) or 1 - (obtained value - minimum value) / (maximum value - minimum value) for each parameter. The final microCT score is the average of the sum of the five parameters after processing.

[0084] Depend on Figure 5 It can be seen that bone destruction was alleviated in rats in the adenovirus Ad-Fbxl16 group, as shown by the scores of five disease-related micro-computed tomography (micro-CT) analysis indicators (including BV, BS, TMD, TMD and micro-CT).

[0085] 3. HE staining and immunofluorescence analysis of pathology and vimentin expression

[0086] The left hind leg fixed in 4% PFA was replaced with fresh fixative and sent to Wuhan Saiweier Biotechnology Co., Ltd. for decalcification, dehydration, embedding, sectioning, HE staining, and storage at room temperature in the dark.

[0087] Immunofluorescence staining involves: ① Dewaxing paraffin sections: Before staining, paraffin sections should be placed at 60°C for 1 hour, immersed in xylene I and II for 10 minutes each, and washed twice with a gradient of ethanol: 100% for 2 minutes, 95% for 2 minutes, 80% for 2 minutes, and 70% for 2 minutes; followed by two washes of 5 minutes in distilled water. ② Antigen retrieval: Submerge the sections in sodium citrate buffer (10mM, pH 6.0), cover the sections, and boil in an autoclave for 3 minutes. After cooling slowly, prepare the antigen retrieval solution (10mM sodium citrate buffer, pH 6.0): Stock solution: Solution A: 29.41g trisodium citrate + 1000mL distilled water; Solution B: 21g citric acid + 1000mL distilled water. ③ BSA blocking: Remove the sections from the staining jar, wipe away any moisture from the back of the sections and surrounding tissue on the front (keeping the tissue moist), then add 5% BSA dropwise and incubate at room temperature for 60 minutes. ④ Primary Antibody Incubation: Remove the blocking solution with filter paper and directly add the primary antibody (FBXL16 Polyclonal Antibody (ThermoFisher, PA5-21094), antibody: PBST = 1:250; Vimentin (Abcam, ab8978), antibody: PBST = 1:250) and incubate on a shaker at 4°C overnight. ⑤ Secondary Antibody Incubation: Recover the primary antibody, wash three times with PBST, and add fluorescent secondary antibodies (Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L), antibody: PBST = 1:250; Alexa Fluor 555-conjugated donkey anti-mouse IgG (H+L), antibody: PBST = 1:250) and incubate at room temperature in the dark for 1-2 hours. ⑥ Recover the secondary antibody, wash three times with PBST, air dry, and mount.

[0088] Depend on Figure 6 As shown in (A) and (B), histopathological analysis of rats in each experimental group demonstrated typical symptoms, including joint swelling, bone destruction, and synovial hyperplasia, confirming the anti-arthritic effect of Fbxl16. Furthermore, immunofluorescence staining of synovial tissue revealed an increased expression of Fbxl16 and a decreased level of vimentin, suggesting that injection of adenovirus Ad-Fbxl16 reduced the proliferation rate of synovial fibroblasts.

[0089] 4. Flow Cytometry Multifactor Detection of Rat Serum

[0090] Vortex the desired beads vigorously for 1 minute to thoroughly mix. Take a serum or plasma sample and dilute it halfway using Assay Buffer (50 μL sample + 50 μL Assay Buffer). Add 25 μL Assay Buffer to each sample tube, 25 μL Mitrix B to each standard tube, 25 μL of each standard to the corresponding standard tube, 25 μL of each sample to the corresponding sample tube, 25 μL of beads to each tube, and 25 μL of detection antibody to each tube. Protect from light, shake the EP tube at 1000 rpm, incubate at room temperature for 2 hours, add 25 μL of SA-PE to each tube, protect from light, shake the EP tube at 1000 rpm, incubate at room temperature for 30 minutes, and centrifuge at 1000 × g for 5 minutes. Carefully aspirate 125 μL of the supernatant using a pipette tip. Add 200 μL 1× Wash Buffer to each tube, vortex the beads, centrifuge at 1000×g for 5 min, discard the supernatant, add 300 μL 1× Wash Buffer to each tube, vortex the beads, transfer the beads to the flow cytometry tube, and prepare for flow cytometry.

[0091] Depend on Figure 6 As shown in (C), a magnetic bead-based multiplex assay kit was used to assess 13 cytokines in rat serum. Ad-Fbxl16 suppressed the protein levels of MCP-1, IL-6, and IL-1β, and increased the expression of the anti-inflammatory cytokine IL-10 in rat serum. These data confirm that Fbxl16 inhibits TNF-α-induced inflammation and improves arthritis in AIA rats.

[0092] Example 3: Fbxl16 Activator - LCB (Licorice Chalcone B)

[0093] 1. Efficient screening of Fbxl16 activators using luciferase assay

[0094] Establish a drug screening cell model to screen the target drug. By constructing a Luciferase reporter plasmid containing the human Fbxl16 promoter sequence (its sequence is shown in SEQ ID NO: 3), and stably and efficiently transfecting the recombinant plasmid into HEK293 cells, a model capable of screening drugs that have the effect of upregulating Fbxl16 promoter expression was established, and the target drug was screened using this model. After amplification, the recombinant plasmid was transiently transfected into HEK293 cells via liposomes, and an appropriate concentration of drug was added for stimulation. After 24 hours, the luciferase content in the cell lysate was detected. For drugs that can increase the luciferase content, further verification was performed at the gene and protein levels of RAFLS cells, and the target drug that can upregulate Fbxl16 expression was obtained.

[0095] Depend on Figure 7It can be seen that after LCB treatment, the mRNA expression level and protein level of Fbxl16 were increased, and the fluorescence intensity was increased, indicating that LCB has the effect of activating Fbxl16.

[0096] 2. LCB inhibits LPS-induced inflammation, invasion and apoptosis of RAFLS cells

[0097] RAFLS cells were treated with 10μM, 20μM, and 40μM LCB for 2 hours and then incubated with LPS or TNF-α for 24 hours. RT-PCR was used to detect the inflammatory factors IL-6 and IL-1β. Similarly, flow cytometry was used to examine the effect of LCB on RAFLS cell apoptosis. The following steps were used: the treated cell supernatant and adherent cells were collected, resuspended in 1mL PBS, and centrifuged at 1000rpm at 4°C for 10 minutes. The cells were resuspended in 500μL 1% Binding Buffer and centrifuged at 1000rpm at 4°C for 10 minutes. The supernatant was discarded and the cells were stained with 100μL 1% Binding Buffer, 2μL PI, and 2μL Annexin V-FITC at room temperature for 30 minutes before detection. The Transwell assay is used to study the invasion and migration of RAFLS cells. The following steps are used: Dilute Matrigel 1:6 (can be diluted directly with serum-free culture medium) and coat the upper chamber of the bottom membrane of a Transwell chamber. Incubate at 37°C for 1-4 hours to allow the Matrigel to polymerize. Add 200 μL of cell suspension to the Transwell chamber, and add 600 μL of culture medium containing 15% FBS to the lower chamber of a 24-well plate. Incubate for 12-48 hours. Remove the Transwell chamber, discard the culture medium, wash twice with calcium-free PBS, and gently wipe away unmigrated cells from the upper layer with a cotton swab. Fix the chamber with methanol or formaldehyde for 30 minutes and air-dry. Stain with 0.1% crystal violet for 30-60 minutes and wash three times with PBS. Gently wipe away moisture from the upper chamber with a cotton swab. To further investigate whether LCB inhibits inflammation by regulating Fbxl16, Fbxl16 was knocked down by siRNA and then LCB was administered for 24 h, and the inflammatory factors IL-6 and IL-1β were detected.

[0098] Depend on Figure 8 It can be seen that in the presence of LPS or TNF-α, the relative mRNA expression levels of IL-6 and IL-1β in RAFLS cells treated with LCB decreased, indicating that LCB can inhibit the expression of inflammatory factors.

[0099] Depend on Figure 9It can be seen that in the presence of LPS, the invasion rate of RASFs cells treated with LCB decreased and the apoptosis rate increased, and LCB achieved an inflammation-inhibiting effect by promoting the expression of Fbxl16, thereby reducing the gene levels of proinflammatory factors IL-1β and IL-6.

[0100] 3. LCB targets Fbxl16 to achieve anti-arthritis effects

[0101] SD rats were purchased and injected intradermally with 100 μL of emulsified oil at the base of the tail. The first inflammation occurred approximately 9 days after adjuvant injection. The paw volume of the rats was measured and recorded every three days. Thirty male rats were randomly divided into five experimental groups: (1) healthy control group (n=6), which did not receive treatment; (2) arthritis model control group (n=6), which was an AIA rat experimental group that received the same excipient as the drug; (3) MTX positive control group (n=6), which was an AIA rat gavage with MTX 7.6 mg / kg / week; (4) low-dose LCB treatment group (n=6), which was an AIA rat gavage with LCB at a dose of 5 mg / kg / day; (5) high-dose LCB treatment group (n=6), which was an AIA rat gavage with LCB at a dose of 10 mg / kg / day. At the end of the treatment course, the rats were sacrificed, and their blood and some organs were collected. The right hind paw was photographed and frozen, and the left hind paw was amputated and fixed in 4% PFA. Erythrocyte sedimentation rate (ESR) was recorded, and gene expression of inflammatory factors IL-6 and IL-1β in the synovial membrane was measured. Rat spleen tissue was washed multiple times with normal saline and then passed through a cell strainer to remove inadequately dispersed cell clusters, tissue fragments, and impurities, thereby rapidly isolating uniform single-cell or multi-cell suspensions. Surface antibody staining was performed with 100 μL cell staining buffer plus 0.5 μL surface antibodies, including CD45, CD3, CD4, and CD8. The suspension was resuspended and incubated at 4°C for 1 hour. The suspension was centrifuged at 2800 rcf for 2 minutes at 4°C, and the supernatant was discarded. The membrane was permeabilized and resuspended in 100 μL True Nuclear Fix Dilution and incubated at 4°C for 1 hour. Cell activation cocktail (with Brefeldin A) was then used for stimulation for 4-8 hours. Inflammatory antibody staining was performed with 100 μL True Nuclear Fix Dilution plus 0.5 μL IL-17, incubated at 4°C for 1 hour, and analyzed by flow cytometry.

[0102] Depend on Figure 10 It can be seen that LCB treatment can significantly reduce the arthritis score and paw swelling volume of AIA rats.

[0103] Depend on Figure 11It was found that in the LCB-fed rat group, the erythrocyte sedimentation rate and the mRNA levels of IL-6 and IL-1β in the synovial tissue were significantly decreased.

[0104] Depend on Figure 12 As shown in Figure A, bone erosion and trabecular number were also improved in all LCB-treated groups, reaching levels comparable to those of the MTX-positive control group, without causing observable toxic effects in other organs. Figure 12 As shown in Figure B, flow cytometry analysis confirmed significantly higher CD3 expression in spleen cells in the AIA model group. However, CD3 levels were reduced in the LCB-treated group compared to the AIA group. No significant changes in CD4 / CD8 levels were observed. In addition, IL-17 levels, which play a key role in the pathogenesis of rheumatoid arthritis, were significantly decreased in the LCB-treated group.

[0105] In summary, the present invention proposes and identifies novel anti-arthritic effects of the Fbxl16 gene in vitro and in vivo. Furthermore, the novel Fbxl16 activator, LCB, has been identified as a potential natural anti-arthritic compound through its immunomodulatory and anti-inflammatory properties, targeting the novel function of FBXL16 in arthritis.

[0106] The embodiments of the present application have been described in detail above with reference to the accompanying drawings. However, the present application is not limited to the above embodiments. Various modifications can be made within the scope of knowledge possessed by ordinary technicians in the relevant technical field without departing from the purpose of the present application. In addition, the embodiments of the present application and the features of the embodiments can be combined with each other unless there is a conflict. SEQUENCE LISTING <110> Macau University of Science and Technology <120> Application of Fbxl16 protein and gene recombinant vector in the preparation of anti-inflammatory drugs <130> 1 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <400> 1 ctgccatctc acaacttcta 20 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <400> 2 acatactcca gtgccatatc 20 <210> 3 <211> 1547 <212> DNA <213> Artificial Sequence <400> 3 cgccgctccc ctgttgcccc aactgtcttc acggggctcg tcctgtctgc cccactccgg 60 gcaccttctt tggtcagtat ccaggtaccg ggcaagactt acgcagaaga gctgctgcgc 120 tcaccccagg tagacccagt ggcgccccca cagcaagcac agaaccaccc caccccggga 180 cccagccagg gtgccgtcag tctgaccctc cttcccgctg accccaggcc tcacaccggc 240 cccttcggcc aggcttccca tcaggacgct gggaacagca gctggtttca ctcctattga 300 gatactctca ggagccccat tcagccctgt agctctttcc cggtgcaaag aacccctctg 360 ctctccccac acctgctccc tcccccggcc ctgctcccag gagaccccag gatagggcgc 420 cattggcctc cattctcctg ccctgagcct gctggggcgt ctggggtctc cagcggtccc 480 caccgagtcc tccacctaac ctagcacgcc ccacccctgc cccgggctgc agcagccgcc 540 gccgcctcca gcctccagtt tattcccgag ggaacttgcc aggctcctcg gatctttgca 600 cccccattcc cgagccatcc ctccctcgcg ccgtgggggg gtctcagctc cagtggggca 660 ccctcaggaa gcccccgcca gaagtcacca ccgcaagccc tcgggtgctt tcactgtcac 720 tgtctgaact gatccgtgca gtcacttgtt ttcgcgctct gcgggtgtgc cagggtctcc 780 aatgaggggc ttccggccgg gcacgcagca ggtgcgcggg gggtgtgagg gctgtgaagg 840 gtccttgccg tgggctgggc gggagggtcc agagacgggg gtctgaccac cctgtctcct 900 ctgggccggg tctcgccttc ctcgcctccc caagtggtgg ccaagggccg ggacgcgcgg 960 tgccgccccg gtccccatag gaccccggac ccctgggcca cccgccggcg cctgctctcc 1020 cgcctcccct cgccctccct tctgcctccc gcgccctccc cgcccgccgc gcgccggggt 1080 ttgttattgt gcgggtgccc gcggcgagcg gggcgggggc gcgtgccggg cctggcgcgt 1140 gctcgtggac gccgtgccgg gagcgcgccg cgcgcggggg ccggggcggg gcgggggcgg 1200 ggctcggcat tctcggcgcc cccgcccggg ctcgcgaacc cggattggct cgccacgccg 1260 ggagcgcgcg agggcgcggc gtttggtgcc gggcgggggg cgcgcgcggc gccggcggcg 1320 gccacggagg agcgcgggg gggcgagggg gcccgcctgg ttccctgcaa 1380 agcggcctta tttatctggg cacagcctca gcctccccgg tggggaggctt ggggcggccg 1440 atcctctccc accggggagc tcctttccgt gcgctgccga gggggcccgg ccaggacggg 1500 acgcggggcg cagggcgcgg ccggggccctg ccggccagtc cagcacc 1547

Claims

1. Use of Fbxl16 protein in the preparation of drugs for treating arthritis.

2. Use of a gene recombinant vector containing the Fbxl16 gene in the preparation of drugs for treating arthritis.

3. Use of an Fbxl16 targeting activator in the preparation of drugs for treating arthritis, characterized in that, the Fbxl16 targeting activator is licochalcone B.

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