CN114796456A - Application of Fbxl16 protein and gene recombinant vector in preparation of anti-inflammatory drugs - Google Patents

Abstract

The invention belongs to the technical field of protein medicines, and discloses application of Fbxl16 protein and a gene recombinant vector in preparation of anti-inflammatory medicines. The invention indicates that the Fbxl16 protein can effectively improve inflammatory conditions, has anti-inflammatory efficacy, and can be used as an anti-inflammatory drug for treating inflammatory diseases such as rheumatoid arthritis. Meanwhile, by constructing a gene recombinant vector containing the Fbxl16 gene, a corresponding gene medicament can be developed. In addition, the invention also provides that licochalcone B (LCB) can be used as an Fbxl16 targeting activator to play a role in enhancing anti-inflammatory efficacy.

Description

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

Technical Field

The invention belongs to the technical field of protein medicines, and particularly relates to application of Fbxl16 protein and a gene recombinant vector in preparation of anti-inflammatory medicines.

Background

RA (rheumatoid arthritis) is one of the most common chronic inflammations, characterized by autoimmunity, severe synovial and systemic inflammation, cell proliferation, soft tissue swelling, stiffness and bone erosion on joints. These inflammations may eventually lead to severe disability and premature death. Despite recent advances in clinical treatment of RA, the toxicity of drug therapy remains a significant challenge. In the current clinic, the treatment method or the combination of treatments used varies depending on the type of arthritis. The most widely used drugs, such as the disease modifying antirheumatic MTX and hydroxychloroquine (placonil), slow or prevent the immune system from attacking the body's own articular tissues. However, long-term systemic administration often leads to drug resistance and to off-target. In addition, physical therapy is also helpful in alleviating the pain of arthritis, and once conservative measures are no longer effective, patients also need to undergo surgical treatment, such as joint repair, fusion, or even replacement.

Therefore, the development of more anti-inflammatory drugs with highly efficient and specific targeting characteristics is crucial for the treatment of inflammatory diseases such as RA (rheumatoid arthritis).

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides the application of the Fbxl16 protein and the gene recombinant vector in preparing anti-inflammatory drugs. The invention indicates that the Fbxl16 protein can effectively improve inflammatory conditions, has anti-inflammatory efficacy, and can be used as an anti-inflammatory drug for treating inflammatory diseases such as rheumatoid arthritis. Meanwhile, a corresponding gene medicine can be developed by constructing a gene recombinant vector containing the Fbxl16 gene. In addition, the invention also provides that licochalcone B (LCB) can be used as an Fbxl16 targeting activator to play a role in enhancing anti-inflammatory efficacy.

The invention provides an application of Fbxl16 protein in preparing anti-inflammatory drugs.

The present invention indicates that mRNA levels of Fbxl16(F-box and leucoine rich repeat protein 16) are significantly down-regulated in the blood of Rheumatoid Arthritis (RA) patients and adjuvant-induced arthritis (AIA) rats compared to healthy individuals. Further experiments show that the Fbxl16 protein can also inhibit the production of proinflammatory cytokines and promote the expression of anti-inflammatory factors, thereby playing an anti-inflammatory role. Therefore, the Fbxl16 protein has outstanding potential for preparing anti-inflammatory drugs.

The invention also provides application of the Fbxl16 protein in preparing a medicine for treating arthritis.

Preferably, the arthritis is rheumatoid arthritis.

The Fbxl16 protein can reduce the foot swelling degree and the arthritis score of an adjuvant-induced arthritis rat model (AIA rat), relieve bone destruction and inhibit synovial hyperplasia, so that the Fbxl16 protein can be used for preparing a medicament for treating rheumatoid arthritis.

The invention also provides application of the gene recombinant vector containing the Fbxl16 gene in preparing anti-inflammatory drugs. The gene recombinant vector is constructed by an expression vector and an Fbxl16 gene. The sequence of the Fbxl16 gene is referenced to sequence number NM-153350 of NCBI. The gene recombinant vector can play an anti-inflammatory role through overexpression of Fbxl16, and has outstanding potential as a gene drug.

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

The invention also provides the application of the gene recombinant vector containing the Fbxl16 gene in preparing a medicament for treating arthritis.

The invention also provides application of the Fbxl16 targeted activator in preparing a medicine for treating arthritis.

Preferably, the Fbxl16 targeting activator is licochalcone B.

Licochalcone BLCB is a kind of chalcone present in licorice root, which is generally considered to have some therapeutic effects on cardioprotection, resistance to Alzheimer's Disease (AD), oxidation and free radicals, and induction of cancer cell apoptosis. The invention indicates that licochalcone B (LCB) can activate and increase the transcription, up-regulation gene and protein level of Fbxl16, so the licochalcone B (LCB) can be used as a targeted activator of Fbxl 16. In addition, licochalcone B can inhibit cell invasion, promote apoptosis, improve inflammation index in synovium, reduce IL-17/CD4 expression in spleen, and has immunoregulatory and anti-inflammatory properties.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides that the gene level of Fbxl16 is remarkably reduced in RA patients and adjuvant-induced arthritis rats, and the overexpression of the Fbxl16 can remarkably inhibit the level of proinflammatory factors secreted by synovial cells induced by LPS, which indicates that the Fbxl16 protein has an anti-inflammatory effect. The invention also provides evidence that injection of a gene recombinant vector comprising the Fbxl16 gene into the knee joint can significantly inhibit adjuvant-induced arthritic rat foot swelling volume, arthritis score and proinflammatory cytokine production. In addition, the invention also successfully discovers that licochalcone B (LCB) can be used as a targeting activator of Fbxl16 and has the functions of activating and increasing the transcription of Fbxl16 and up-regulating gene expression and protein level.

Drawings

FIG. 1 is a normalized expression scattergram of each gene in blood of RA patients (A) and AIA rats (B).

FIG. 2 shows mRNA (A) and protein levels (B) of Fbxl16 in RAFLS cells treated with TNF- α, transcriptional activity of Fbxl16 in the presence of LPS, Pam3, poly (I: C), IL-1 β and TNF- α stimulation and in the presence of TNF- α, transcriptional activity of Lef, Chlo, Tet or Nab addition.

FIG. 3 is the expression of inflammatory factors in MH7A cells overexpressing Fbxl16 in the presence of TNF- α.

FIG. 4 is the arthritis score (A) and the paw swelling volume (B) of the AIA rat model after treatment with adenovirus Ad-Fbxl 16.

FIG. 5 is a photograph of Micro-CT radiographs (A) and BV, BS, TMD and Micro-CT scores (B) in a rat model of AIA treated with adenovirus Ad-Fbxl 16.

FIG. 6 shows H & E staining of sagittal section (A), immunofluorescence image (B) of synovial tissue, and serum cytokine content (C) of knee joint of rats in each test group.

FIG. 7 shows the mRNA (A) and protein levels (B) of Fbxl16 in RAFLS cells treated with LCB, and the transcriptional activity (C) of Fbxl16 in HEK293 cells.

FIG. 8 is a graph showing the relative mRNA expression levels of IL-6 and IL-1. beta. in RAFLS cells in the presence of LPS or TNF-. alpha..

FIG. 9 shows the invasion and apoptosis of the RASFs cells treated with LCB in the presence of LPS (A), and the expression levels of IL-1. beta. and IL-6 by knocking out Fbxl16 and LCB (B).

FIG. 10 is the arthritis score (A) and foot swelling volume (B) of AIA rats after oral LCB administration.

FIG. 11 shows erythrocyte sedimentation rate (A) and expression amounts (B) of IL-6 and IL-1. beta. in synovial tissue of AIA rats after oral administration of LCB.

FIG. 12 is a Micro-CT radiographic image (A) of bone erosion and trabecular bone in LCB-treated rats, an immune-related index of spleen single cells (B).

Detailed Description

In order to make the technical solutions of the present invention more apparent to 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 the claimed protection scope is not limited thereto, and any modification, substitution, combination made without departing from the spirit and principle of the present invention are included in the protection scope of the present invention.

The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.

Example 1: anti-inflammatory action of Fbxl16 protein

1. Blood sample total RNA extraction

Blood samples were collected from RA patients and healthy volunteers at the general guangdong hospital, the medical science institute of guangdong province, guangzhou (china) with informed and voluntary consent signed and approved by the research ethics committee (GDREC 2015391H). All epidemiological investigations and classifications by volunteers were performed according to the standards of the american college of rheumatology. Wild type male Sprague-Dawley (SD) rats weighing 80-120g were purchased from the laboratory animal services center, university of Chinese, hong Kong, China. The animal care and handling procedures were in compliance with institutional guidelines and the regulations of animal regulations (special administrative department health agency of hong kong, china).

Total RNA from Blood was isolated using the FavorPrepTM Blood/pooled Cell Total RNA mini kit (Favorgen Biotech). 200-L anticoagulant-preserved fresh whole blood, 5 volumes of RL buffer was mixed with 1 volume of sample, inverted and stirred well before incubation on ice for 10 minutes with brief 2 vortexes. The cell pellet was formed by centrifugation at 4500rpm for 1 minute. Discard the supernatant, add 600. mu.L RL buffer, briefly vortex to resuspend the cell pellet, centrifuge at 4500rpm for 1min, form the cell pellet again, and discard the supernatant completely. mu.L FARB buffer and 3.5. mu.L beta-mercaptoethanol were added to the cell pellet and vortexed vigorously for 1 minute to completely resuspend the cells. The filter column was placed in the collection tube and the sample mixture was transferred to the filter column and centrifuged at maximum speed for 2 minutes. The clarified supernatant was transferred from the collection tube to a new microcentrifuge tube, and the volume of the supernatant was measured, 1 volume of 70% ethanol was added, and vortexed to mix well. The FARB column was placed in the collection tube, the ethanol-added sample mixture (including any precipitate) was transferred to the FARB column, centrifuged at full speed for 1 minute, the flow-through sample discarded, and the FARB column was replaced. The FARB column was returned to the collection tube, 500. mu.L of wash buffer 1 was added to the FARB column, centrifuged at full speed for 1 minute, the liquid that passed through was discarded, and the FARB column was returned to the collection tube. Add 750. mu.L wash buffer 2 to the FARB column, centrifuge at full speed for 1 minute, discard the liquid that flows through, place the FARB mini-column back into the collection tube, repeat 1 time. The FARB column was centrifuged at full speed for 3 minutes. Placing FARB column into elution tube, adding 40-100 μ L RNase-free ddH into the center of FARB column membrane ₂ And O. UV spectrophotometry (NanoDrop Technologies, USA) was used to measure RNA quality and concentration. Mu.g of RNA was reverse transcribed with Maxima H Minus cDNA Synthesis Master Mix (Thermo, USA). The reaction was terminated by incubating the mixture at 25 ℃ for 10 minutes, then at 50 ℃ for 15 minutes and heating at 85 ℃ for 5 minutes. mu.L of PCR Mix consisted of 0.5. mu.L of template, 0.5. mu. L F primer (5'-CTGCCATCTCACAACTTCTA-3' (SEQ ID NO: 1)) and R primer (5'-ACATACTCCAGTGCCATATC-3' (SEQ ID NO: 2)), 0.5. mu.L of template, 10. mu.L of SYBR Master Mix, and 8.5. mu.L of ddH ₂ O, 20 μ L in total. Quantification of gene expression was performed by a ViiA 7 real-time PCR System (Applied Biosystems). All mRNA data were normalized by the reference gene β -actin and compared using the Δ Δ CT methodAnd (4) quantifying.

FIG. 1 is a normalized expression scatter plot of each gene in blood of RA patients (as shown in FIG. 1 (A)) and AIA rats (as shown in FIG. 1 (B)), with the centerline indicating unaltered gene expression, the dashed line indicating a 3-fold regulatory threshold, and the data points outside the dashed line and in the lower right portion corresponding to the selected regulatory threshold. The results in fig. 1 show that the mRNA level of Fbxl16 was significantly down-regulated in the blood of both RA patients and AIA (adjuvant-induced arthritis) rats compared to healthy controls.

TNF-alpha induced reduction of Fbxl16 gene, protein, and transcript levels

RASFs (rheumatoid arthritis synovial fibroblasts) cells were seeded in 6-well plates, stimulated with 10 and 20 ng/mL TNF-alpha for 24 hours the next day, and gene and protein expression levels were detected using Fbxl 16-specific primers (F primer: 5'-CTGCCATCTCACAAC TTCTA-3' (SEQ ID NO: 1); R primer: 5'-ACATACTCCAGTGCCATATC-3' (SEQ ID NO: 2)) and Antibody (FBXL16 polyclone Antibody (ThermoFisher, PA 5-21094)). The samples were lysed with Protein extraction buffer (ab156034) on ice for 15 minutes, centrifuged at 13000g for 15 minutes at 4 ℃, and after collecting the supernatant and quantitating it with Protein Assay Dye Reagent (Bio-Rad), the concentration of these proteins was measured using a spectrophotometer at 595 μm. Mu.g of protein samples were detected by 12% SDS-PAGE and transferred to PVDF membranes (Bio-Rad) which were blocked with 5% BSA for 1 hour, the membranes were bound with anti-Fbxl 16 antibody (ThermoFisher; antibody: PBST ═ 1:1000) and anti-GAPDH antibody (Santa Cruz Biotechnology, antibody: PBST ═ 1:2000) overnight at 4 ℃ and, after washing with TBST, incubated with anti-rabbit and anti-mouse secondary antibodies (Santa Cruz Biotechnology, antibody: PBST ═ 1:2000) for 2 hours at room temperature. The hypersensitivity ECL chemiluminescence liquid kit (Beijing 4A Biotec h co., Ltd) was used to detect bands under the Amersham Imager 600(GE) imaging system, and the intensity of the bands was analyzed using ImageJ software.

Construction of pEZX-PL01-Fbxl16 plasmid

pHBAAV-CMV-MCS-3flag-T2A-ZsGreen plasmid is used as a gene vector, each reagent is sequentially added according to the sequence in the table 1, the mixture is lightly sucked, uniformly mixed and placed in a water bath kettle at 37 ℃ for reaction for 1 to 2 hours; after the enzyme digestion is finished, carrying out agarose gel electrophoresis, and recovering a linearized vector;

TABLE 1 vector cleavage System

PCR amplification of target gene fragment

The amplification system is prepared by adopting the contents in the table 2, the mixture is gently mixed, the mixture is placed in a PCR instrument to be amplified by adopting the PCR program shown in the table 3, and a target gene fragment (Fbxl16 gene, the sequence of the Fbxl16 gene refers to the sequence number NM-153350 of NCBI) is obtained through amplification.

TABLE 2 PCR amplification System

TABLE 3 PCR procedure

Connecting the target gene segment with a linearized vector

The following reaction system of table 4 was prepared in an ice-water bath, reacted at 50 ℃ for 30min, and then placed on ice for 5min to obtain a ligation product, which was immediately subjected to subsequent transformation.

TABLE 4 reaction System

Transformation of

1) Taking out DH5 alpha competent cells from a refrigerator at the temperature of-80 ℃, immediately putting the cells on ice for melting, and gently operating the competent subpackaging process to reduce the mechanical damage to the cells;

2) after the competence is melted, subpackaging by 50 mu L of each tube, adding the ligation product by an amount not exceeding the competence volume 1/10 after subpackaging, and standing on ice for 20-30 min;

3) performing heat shock at 42 deg.C for 90s, immediately inserting into ice for ice breeding for 2-3 min;

adding 500. mu.L LB medium into the super clean bench, and slightly inverting 3-5 times;

5) shaking and culturing at 37 deg.C and 230rpm for 45-60 min;

6) and (3) coating the bacterial liquid on a solid plate with corresponding resistance, uniformly coating, and then inversely placing the plate in a 37 ℃ incubator for culturing for 12-16h to complete the construction of the pEZX-PL01-Fbxl16 plasmid.

Using liposomes (

3000, Invitrogen) transfection reagent the pEZX-PL01-Fbxl16 recombinant plasmid was transferred into HEK293 cells and seeded in 24-well plates at 2X 10 per HEK293 cell ⁵ And (4) respectively. The treatment is carried out according to the following 2 ways: add LPS, TNF-. alpha., IL-1. beta., Pam3CSK4(Pam3) and poly (I: C) HMW to each well and incubate the cells at 37 ℃ for 24 hours. ② after 2 hours of pretreatment of leflunomide (Lef), chlortetracycline (Chlo), tetracycline (Tet) and nalbupone (Nab), TNF-alpha is added and incubation is continued for 24 hours. Removing the culture medium, washing the cultured cells in 1 XPBS, removing all washing liquid, adding 1 XPLB, cracking the cells for 15min at room temperature, adding 10 mu L of cell lysate into 100 mu L of LAR II, uniformly blowing, and detecting the reading, namely the value of Fireflyfluenase. Add 100. mu.L of Stop&Glo, again read, the value for Renilla luciferase. Data processing: firstly, the ratio of Firefly luciferase/Renilla luciferase of each tube is calculated, and then the ratio of the control group is taken as a unit 1, so that the relative luciferase activity of different treatment groups, namely the regulation and control activity of gene transcription of the treatment groups can be obtained.

As shown in FIGS. 2 (A) and (B), the levels of mRNA and protein of Fbxl16 were significantly reduced in RAFLS (rheumatoid arthritis fibroblast-like synovial cells) cells under TNF-. alpha.stimulation. Dual luciferase reporter experiments demonstrated 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-. alpha.and IL-1. beta.). In contrast, as shown in FIG. 2 (C), the transcriptional activity of Fbxl16 was enhanced in the presence of TNF- α with the addition of DMARDs or anti-inflammatory drugs such as leflunomide (Lef), chlortetracycline (Chlo), tetracycline (Tet), and nalbupone (Nab).

3. Real-time quantitative PCR detection of inhibition of inflammatory factor expression after over-expression of Fbxl16

MH7A cells (2X 10) ⁶ Individual cells/well) were seeded onto 6-well plates and cultured overnight to allow attachment. Using liposomes (

3000, Invitrogen) transfection reagent the pEZX-PL01-Fbxl16 recombinant plasmid was transferred into MH7A cells, incubated at 37 ℃ for 24 hours, and cultured with TNF-. alpha.in a 6-well plate for 24 hours. Total RNA was extracted using the FavorPrep Total RNA purification mini kit (Favorgen, PingTung, Taiwan),

VILO Master Mix (Invitrogen, Scotland, UK) was used to synthesize cDNA by reverse transcription, and real-time quantitative PCR was used to detect the expression levels of IL-6, IL-1. beta. and TNF-. alpha.genes, inflammatory factors.

As shown in FIG. 3, after overexpression of Fbxl16, a reduction in IL-6 and TNF-. alpha.mRNA levels was observed in TNF-. alpha.induced MH7A cells, confirming the anti-inflammatory effect of Fbxl16 protein.

Example 2: overexpression of Fbxl16 can inhibit inflammatory response and bone destruction in AIA rats

1. Achieving adenovirus-mediated high expression of Fbxl16 in AIA rat model

Male SD rats of 4-6 weeks old, purchased from Guangdong province medical laboratory animal center, weighing 80-120g, were used in this experiment. The animals were housed in a room equipped with a temperature control and automatic ventilation system, with 12 hours light/dark cycle, and were allowed to eat and drink at will. The study was approved by the animal ethics committee of the department of health in the special area of australia, china, and was conducted according to the guidelines of the animal care and user committee of the university of australia science and technology. Mineral oil (Sigma, USA) containing 2.5mg/mL of inactive Mycobacterium tuberculosis was ground for a long time to emulsify complete Freund's adjuvant, and 100. mu.L of emulsified oil was injected into rat tail root. The first inflammation appeared about day 9 after adjuvant injection, and rat paw volumes were measured and recorded every three days to construct a rat model of adjuvant-induced arthritis (AIA rats).

32 male rats were taken and randomly divided into 5 experimental groups: (1) healthy rat control group (n ═ 6), no treatment; (2) adjuvant-induced arthritis model control group (n ═ 6); (3) MTX (methotrexate) positive control (n ═ 6), gavage MTX 7.6mg/kg/week in AIA rats; (4) adenovirus Ad-GFP (green fluorescent protein) group (n ═ 6), 100 μ L of adenovirus Ad-GFP was injected into the joint cavity of AIA rats using a microinjector; (5) adenovirus Ad-Fbxl16 group (n 8), 100 μ L of adenovirus Ad-Fbxl16 was injected into the joint cavities of AIA rats using a microinjector. After the test groups are set, continuously observing every day to obtain the arthritis score and the foot swelling volume information of each test group. At the end of the treatment period, the rats were sacrificed and blood and part of the visceral organs were collected, the right hind paw was photographed and cryopreserved, and the left hind paw was amputated and fixed in 4% PFA.

The main construction methods of the adenovirus Ad-GFP and the adenovirus Ad-Fbxl16 are as follows:

1) the first day: AAV-293 cells were passaged into 100mm dishes for transfection. After the operation is finished, the mixture is placed at 37 ℃ and 5 percent CO ₂ And 95% relative humidity.

2) And on the third day: transfection is performed until the cell density reaches about 80-90% confluence.

And (3) carrying out lipofection: opti MEM was preheated in a 37 ℃ water bath, and the Lipofeter transfection reagent was returned to room temperature before use, and shaken well before use. The transfection complex composition is shown in table 5 below.

TABLE 5 transfection complexes

3) Liquid changing: fresh complete medium containing 10% fetal bovine serum FBS was replaced 6h after transfection.

4) Cell collection: 72h after transfection, cells containing AAV particles were gently scraped with a cell scraper, collected in a 15mL centrifuge tube, centrifuged at 150 Xg for 3min to collect cells, culture supernatant was removed, washed once with PBS, and finally resuspended in 300. mu.L PBS.

5) Cell disruption: preparing a constant-temperature water bath kettle at 37 ℃ and liquid nitrogen, and repeatedly freezing and thawing the centrifuge tube filled with the cells in the liquid nitrogen and the water bath at 37 ℃ for three times. The cells were centrifuged at 2000 Xg for 5min at 4 ℃ to remove cell debris, and the lysate supernatant containing AAV particles was collected.

6) And (3) purification: 0.1 mu.L of Benonase enzyme is added into each 1mL of the lysis supernatant, and water bath is carried out at 37 ℃ for 1h, so as to remove cell genome and residual plasmid DNA in virus solution. Centrifuging at 600 Xg and 4 deg.C for 10min, and collecting supernatant. Column purification was performed according to the Biomiga adeno-associated virus purification kit V1469-01. 4mL of AAV virus sample liquid obtained by column purification was added to an ultrafiltration tube, and centrifuged at 1400 Xg for 30min to obtain about 200. mu.L of AAV. The purified virus obtained finally was collected and stored at-80 ℃.

As can be seen from FIG. 4, the anti-inflammatory effect of Fbxl16 protein was further verified by injecting adenovirus Ad-Fbxl16 into the knee joint of AIA rats. Although AIA rats injected with adenovirus Ad-GFP showed joint stiffness and severe swelling of the feet, Ad-Fbxl16 (1X 10) ¹¹ PFU/mL) rats had significantly reduced arthritis scores and swollen volume in the feet.

2. Micro computer tomography (MicroCT) analysis of rat foot bone destruction

The right hind paw was thawed on ice and a micro-CT scanner (SkyScan1176, Bruker, Belgium) was used for the right hind paw. The scan parameters were at 35 micron resolution, 62kV, 385 μ A, 98ms exposure time, angular velocity 0.70, and Al 1mm filter. After scanning is finished, images are reconstructed by using NReco software (Bruker-micro CT), CTvox software is used for opening reconstructed data files and generating observable three-dimensional pictures, and CTAn software is used for analyzing scanning data.

MicroCT scores are derived from five disease-related indicators including bone mineral density, bone volume fraction, cortical mineral density, trabecular number, and total porosity as examined by micro-computed tomography MicroCT. The formula for calculating the MicroCT score is as follows: (get-min)/(max-min) or 1- (get-min)/(max-min) for each index. And finally, the MicroCT score is the average value of the sum of the five indexes after the treatment.

As can be seen in FIG. 5, the adenoviral Ad-Fbxl16 group rats exhibited reduced bone destruction as indicated by the scores of the 5 disease-related micro computerized tomography (micro-CT) analysis indices, including BV, BS, TMD and micro-CT.

HE staining and immunofluorescence analysis of pathology and vimentin expression

The left hind paw fixed with 4% PFA was replaced with fresh fixative, sent to Wuhan Seville Biotech Ltd for decalcification, dehydration, embedding, sectioning, HE staining, and kept at room temperature and in dark for future use.

Immunofluorescence comprises the steps of paraffin section dewaxing: paraffin sections should be placed at 60 ℃ for 1 hour before staining, and xylene I, II should be soaked for 10 minutes each, gradient alcohol: 100%, 2 minutes; 95%, 2 minutes; 80%, 2 minutes; 70%, 2 minutes; washing with distilled water: 5min, 2 times. Antigen retrieval: sodium citrate buffer (10mM, pH6.0), submerging slices, covering the pot, boiling in an autoclave, slowly cooling after 3 minutes, preparing antigen retrieval solution (10mM, pH6.0 sodium citrate buffer): preparing a stock solution: solution A: 29.41g of trisodium citrate and 1000mL of distilled water; and B, liquid B: 21g of citric acid and 1000mL of distilled water; ③ BSA blocking: the sections were taken out from the staining jar, wiped to remove water on the back of the sections and water around the tissues on the front of the sections (keeping the tissues in a wet state), 5% BSA was added dropwise, and treated at room temperature for 60 minutes. Incubation primary antibody: the blocking solution was aspirated through a filter paper, and the first Antibody (FBXL16 Polyclonal Antibody (ThermoFisher, PA5-21094), PBST 1:250, Vimentin (Abcam, ab 16), PBST 1:250, was added directly dropwise and shaken overnight at 4 ℃. Incubating a second antibody: the primary antibody was recovered, washed 3 times with PBST, and a fluorescent secondary antibody (Alexa Fluor 488-labeled goat anti-rabbit IgG (H + L) antibody: PBST ═ 1: 250; Alexa Fluor 555-labeled donkey anti-mouse IgG (H + L) antibody: PBST ═ 1:250) was added dropwise thereto, followed by incubation at room temperature in the dark for 1-2 hours. Sixthly, recovering the secondary antibody, washing for 3 times by PBST, drying and sealing.

As can be seen from (A) and (B) in FIG. 6, the anti-arthritic effect of Fbxl16 was confirmed by evaluating typical symptoms of joint tissue swelling, bone destruction, synovial hyperplasia, etc. in rats of each test group by histopathological analysis. In addition, immunofluorescent staining of synovial tissue showed an increased tendency for Fbxl16 expression, but decreased levels of vimentin, indicating that injection of adenovirus Ad-Fbxl16 reduced the proliferation rate of synovial fibroblasts.

4. Flow-through multi-factor assay for rat serum

The desired Beads were vortexed vigorously for 1min to mix well. Serum plasma samples were taken and diluted one-fold using Assay buffer (50. mu.L sample + 50. mu.L Assay buffer). Add 25. mu.L Assay Buffer to each sample tube, 25. mu.L Mitrix B to each standard tube, 25. mu.L each standard to the corresponding standard tube, 25. mu.L each sample to the corresponding sample tube, 25. mu.L beads to each tube, and 25. mu.L detection antibody to each tube. Shaking the EP tube at 1000rpm in a dark place, incubating at room temperature for 2h, adding 25 μ L of SA-PE to each tube in a dark place; the EP tube was shaken at 1000rpm, left at room temperature for 30min, and centrifuged at 1000 Xg for 5 min. The supernatant 125. mu.L was carefully aspirated off with a pipette tip. Add 200. mu.L of 1 × Wash Buffer per tube, vortex Beads, centrifuge for 5min at 1000 × g, discard supernatant, add 300. mu.L of 1 × Wash Buffer per tube, vortex Beads, transfer Beads to flow tube, ready for flow-on-machine.

As can be seen from FIG. 6 (C), 13 cytokines were evaluated in rat serum using a magnetic bead-based multiplex assay kit, and it was found that adenovirus Ad-Fbxl16 inhibited the protein levels of MCP-1, IL-6 and IL-1 β and increased the expression level of anti-inflammatory factor IL-10 in rat serum. These data demonstrate that Fbxl16 inhibits TNF- α induced inflammation, improving the arthritic status of AIA rats.

Example 3: fbxl16 activator-LCB (Licorice chalcone B)

1. Efficient screening of Fbxl16 activator by luciferase experiment

And establishing a drug screening cell model and screening the target drug. A model capable of screening out drugs with an up-regulated Fbxl16 promoter expression effect is established by constructing a Luciferase report plasmid containing a human Fbxl16 promoter sequence (the sequence of the Luciferase report plasmid is shown as SEQ ID NO: 3), stably and efficiently transfecting the recombinant plasmid into an HEK293 cell, and a target drug is screened out by utilizing the model. The recombinant plasmid is amplified and then transiently transfected into HEK293 cells through liposome, drugs with proper concentration are added for stimulation, and the content of luciferase in cell lysate is detected after 24 hours. And (3) continuously verifying the gene and protein levels of the RAFLS cells for the medicine capable of increasing the luciferase content, so as to obtain the target medicine capable of up-regulating the expression of Fbxl 16.

As can be seen from FIG. 7, the mRNA expression level and protein level of Fbxl16 were increased and the fluorescence intensity was increased after LCB treatment, indicating that LCB has the effect of activating Fbxl 16.

LCB inhibition of LPS-induced RAFLS cell inflammation, invasion and apoptosis

After the RAFLS cells are treated by LCB with 10 mu M, 20 mu M and 40 mu M respectively for 2h, the cells are incubated with LPS or TNF-alpha for 24h, and inflammatory factors IL-6 and IL-1 beta are detected by RT-PCR experiments. In the same way, the flow cytometry is used for detecting the influence of LCB on the RAFLS cell apoptosis, and the specific steps are as follows: collecting the treated cell supernatant and adherent cells, resuspending in 1mL PBS, centrifuging at 1000rpm and 4 ℃ for 10min, resuspending the cells in 500 μ L1 Binding Buffer, centrifuging at 1000rpm and 4 ℃ for 10min, discarding the supernatant, staining with 100 μ L1 Binding Buffer +2 μ L PI +2 μ L Annexin V-FITC at room temperature for 30min, and detecting on a machine. The Transwell experiment is used for researching the invasion and migration condition of the RAFLS cells, and comprises the following specific steps: using a Matrigel 1: 6 dilution (can be directly diluted with serum-free medium), coating the upper chamber surface of the bottom membrane of the Transwell chamber, and placing in an incubator at 37 ℃ for 1-4h to polymerize Matrigel into gel. 200. mu.L of the cell suspension was added to the Transwell chamber, and 600. mu.L of the medium containing 15% FBS was added to the lower chamber of the 24-well plate, and the cells were cultured for 12 to 48 hours as usual. The Transwell chamber was removed, the medium from the wells was discarded, washed 2 times with calcium-free PBS, the upper non-migrated cells were gently wiped off with a cotton swab, fixed with methanol or formaldehyde for 30 minutes, and the chamber was appropriately air-dried. Stained with 0.1% crystal violet for 30-60min and washed 3 times with PBS. The upper chamber was gently wiped with a cotton swab. To further investigate whether LCB inhibits inflammation by modulating Fbxl16, the inflammatory factors IL-6 and IL-1 β were detected by siRNA knockdown of Fbxl16 followed by administration of LCB for 24h treatment.

As can be seen in FIG. 8, the relative mRNA expression levels of IL-6 and IL-1. beta. in the presence of LPS or TNF-. alpha.in the RAFLS cells treated with LCB were decreased, indicating that LCB could inhibit the expression of inflammatory factors.

As can be seen from fig. 9, in the presence of LPS, the rates of invasion and apoptosis of the RASFs cells treated with LCB decreased, and LCB reduced the gene levels of the pro-inflammatory factors IL-1 β and IL-6 by promoting the expression of Fbxl16 to inhibit inflammation.

LCB targeting Fbxl16 for anti-arthritic effects

SD rats were purchased and 100. mu.L of emulsified oil was injected intradermally into the tail roots. The first inflammation appeared about day 9 after adjuvant injection. Rat paw volumes were measured and recorded every three days. 30 male rats were taken and randomly divided into 5 experimental groups: (1) healthy control group (n ═ 6), no treatment; (2) arthritis model control group (n ═ 6), AIA rat experimental group received the same vehicle as the drug; (3) MTX positive control group (n ═ 6), AIA rats were gavaged with MTX 7.6 mg/kg/Week; (4) low dose LCB treatment group (n ═ 6), at a dose of 5mg/kg/Day, the lca rats were gavaged with LCB; (5) high dose LCB treatment group (n ═ 6), the lca rats were gavaged with LCB at a dose of 10 mg/kg/Day. At the end of the treatment period, the rats were sacrificed, blood and part of the visceral organs were collected, the right hind paw was photographed and cryopreserved, and the left hind paw was amputated and fixed in 4% PFA. Recording blood sedimentation, and detecting the expression quantity of inflammatory factors IL-6 and IL-1 beta genes in the joint synovium. After being washed by normal saline for a plurality of times, spleen tissues of rats are passed through a cell filter to remove cell masses, tissue blocks or impurities which are not fully dispersed, so that uniform single-cell or multi-cell suspensions are rapidly separated, and surface antibodies stain 100 mu L of cell stabilizing buffer +0.5 mu L of surface antibodies, including CD45, CD3, CD4 and CD 8; resuspending at 4 ℃ for 1h, 2800rcf, centrifuging at 4 ℃ for 2min, discarding the supernatant, disrupting 100. mu.L of True Nuclear Fix Dilution, resuspending at 4 ℃ for 1h, stimulating with cell activation cocktail (with Brefeldin A) for 4-8h, staining with inflammatory antibody for 100. mu.L of True Nuclear Fix Dilution + 0.5. mu.L of IL-17 for resuspension, incubating at 4 ℃ for 1h, and detecting with a flow cytometer.

As can be seen in fig. 10, treatment with LCB resulted in a significant reduction in both the arthritis score and the volume of foot swelling in the AIA rats.

As can be seen from FIG. 11, in the group of LCB-fed rats, erythrocyte sedimentation rate and mRNA levels of IL-6 and IL-1. beta. in synovial tissue were significantly reduced.

As can be seen in fig. 12, bone erosion and trabecular number were also improved in all LCB-treated groups to levels comparable to MTX positive control groups, without causing observable toxic effects in other organs. As can be seen from B in fig. 12, the expression of CD3 was significantly higher in spleen cells of the AIA model group as confirmed by flow cytometry analysis. However, the level of CD3 was reduced in the LCB-administered group compared to the AIA group. No significant change in the level of CD4/CD8 was observed. In addition, IL-17 levels, which play an important role in the pathogenesis of rheumatoid arthritis, were significantly reduced in the LCB-administered group.

In conclusion, the present invention proposes and determines a novel anti-arthritic effect of the Fbxl16 gene in vitro and in vivo. Furthermore, the new Fbxl16 activator LCB was identified as a potential anti-arthritic natural compound by its immunomodulatory and anti-inflammatory properties, against the new function of Fbxl16 in arthritis.

The embodiments of the present application have been described in detail with reference to the drawings, but the present application is not limited to the embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present application. Furthermore, the embodiments and features of the embodiments of the present application may be combined with each other without conflict.

SEQUENCE LISTING

<110> Australian university of science and technology

<120> Fbxl16 protein and application of gene recombinant vector in 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

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<211> 20

<212> DNA

<213> Artificial sequence

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acatactcca gtgccatatc 20

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<211> 1547

<212> DNA

<213> Artificial sequence

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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 agcgcgggga gggcgagggg aggaggaggc gcccgcctgg ttccctgcaa 1380

agcggcctta tttatctggg cacagcctca gcctccccgg tgggaggctt ggggcggccg 1440

atcctctccc accggggagc tcctttccgt gcgctgccga gggggcccgg ccaggacggg 1500

acgcggggcg cagggcgcgg ccgggccctg ccggccagtc cagcacc 1547

Claims (6)

1. Application of Fbxl16 protein in preparing anti-inflammatory drugs.
2. The application of Fbxl16 protein in preparing a medicine for treating arthritis.
3. 3. The application of the gene recombinant vector containing the Fbxl16 gene in preparing anti-inflammatory drugs.
4. 4. The application of the gene recombinant vector containing Fbxl16 gene in preparing the medicine for treating arthritis.
5. Use of an Fbxl16 targeting activator in the preparation of a medicament for the treatment of arthritis.
6. 6. The use of claim 5, wherein the Fbxl16 targeting activator is licochalcone B.

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