Application of STUB1 protein as BP-3 target in osteoarthritis

Abstract

The invention discloses application of STUB1 protein as a BP-3 target spot in osteoarthritis, and relates to the technical field of medicines. The targeted protein STUB1 of the environmental pollution factor BP-3 induced osteoarthritis is screened, and Bp-3 can be strongly combined with the STUB1, so that the occurrence and progress of the osteoarthritis are induced. The STUB1 gene is knocked out and then BP-3 is used for counteracting toxic substances, compared with a group which is not knocked out and only BP-3 counteracting toxic substances, the bone joint injury index MMP13 is reduced, and the human collagen type 2 Collagen II maintaining joint health is increased, which indicates that the knockout of the STUB1 gene or inhibition of STUB1 protein activity in BP-3-induced osteoarthritis is beneficial to relieving cartilage cell injury.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and more particularly to the application of STUB1 protein as a BP-3 target in osteoarthritis. Background Art

[0002] Osteoarthritis (OA) is a chronic progressive joint disease centered on the degenerative lesion of articular cartilage. Its main features include articular cartilage destruction, subchondral bone sclerosis, osteophyte formation, and synovial inflammatory reaction, ultimately leading to joint dysfunction and pain. Its onset is closely related to factors such as age, obesity, trauma, and genetics. It is commonly found in weight-bearing joints such as the knee joint and hip joint, and is one of the main causes of disability in the middle-aged and elderly population. In 2019, there were approximately 530 million osteoarthritis patients globally, and the age-standardized incidence rate was 492.2 / 100,000. The incidence rate in China (509.8 / 100,000) is higher than the Asian average. In China, with the aging of the population and the change of lifestyle, the prevalence of osteoarthritis shows a rapid upward trend and a trend of getting younger. This trend not only seriously affects the quality of life of patients, but also brings a heavy burden to society and the medical system.

[0003] In recent years, studies have found that environmental pollutants may play an important role in the occurrence and progression of osteoarthritis. Endocrine Disrupting Chemicals (EDCs), as a type of environmental pollutants, have been proven to affect bone health through various mechanisms. Among them, benzophenone-3 (BP-3, Oxybenzone), a widely used ultraviolet absorber, has become one of the potential environmental pollutants in recent years. BP-3 is widely used in sunscreen, cosmetics, and plastic products to provide ultraviolet protection. Through the analysis of population data, we found a significant association between BP-3 exposure and the risk of developing osteoarthritis. However, the pathogenic mechanism of BP-3 in the human body, including the binding target, is still unclear. If the binding target of BP-3 can be screened out, it will not only help to reveal the disease mechanism, but also provide a new direction for the early prevention and intervention of osteoarthritis. Summary of the Invention

[0004] In view of this, the present invention provides the application of STUB1 protein as a BP-3 target in osteoarthritis.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] The application of STUB1 protein as a BP-3 target in osteoarthritis.

[0007] Preferably, regulating the expression of STUB1 protein is used for screening diagnostic reagents for osteoarthritis or screening drugs for alleviating osteoarthritis.

[0008] Another object of the present invention is to provide the use of a reagent for detecting the expression level of STUB1 gene in the preparation of an osteoarthritis diagnostic product.

[0009] Preferably, the reagent for detecting the expression level of STUB1 gene is a kit, and the kit includes a primer set such as SEQ ID NO.1 - SEQ ID NO.4.

[0010] Another object of the present invention is to provide the use of a reagent for detecting the expression level of STUB1 protein in the preparation of an osteoarthritis diagnostic product.

[0011] Preferably, the reagent for detecting the expression level of STUB1 protein is a kit, and the kit includes a STUB1 protein standard.

[0012] Another object of the present invention is to provide the use of a STUB1 protein inhibitor in the preparation of a drug for alleviating osteoarthritis caused by BP-3.

[0013] Beneficial effects:

[0014] The present invention screened the target protein STUB1 for osteoarthritis induced by the environmental pollutant factor BP-3. BP-3 can strongly bind to STUB1, thereby inducing the occurrence and progression of osteoarthritis. By knocking out the STUB1 gene and then challenging with BP-3, compared with the group that was only challenged with BP-3 without knocking out the gene, the osteoarthritis factor MMP13 decreased while the human collagen type II (Collagen II) that maintains joint health increased, indicating that knocking out the STUB1 gene or inhibiting the activity of STUB1 protein in BP-3-induced osteoarthritis helps to alleviate chondrocyte damage. Description of the drawings

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0016] Figure 1 It is a schematic diagram of the docking result of BP-3 and STUB1 molecules.

[0017] Figure 2 It is a schematic diagram of the Western Blot result of the increase in the expression level of STUB1 induced by BP-3.

[0018] Figure 3Schematic diagram of the result that BP-3 induces an increase in the expression level of STUB1 gene.

[0019] Figure 4 Schematic diagram of the Western Blot result that knocking down the expression level of STUB1 gene alleviates Bp-3-induced damage of C28 / I2 chondrocytes. Detailed implementation manners

[0020] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0021] Example 1

[0022] 1. Use the SILAC-SM-PD technology platform of Spectrum Technology (Shanghai) Co., Ltd. to screen and discover the specific target protein of Oxybenzone in cells.

[0023] (1) Use chemical synthesis technology to modify Oxybenzone (Small Molecule, SM) with alkyne (Oxybenzone-alkyne) to prepare a pull-down (PD) probe (Probe).

[0024] (2) Incubate the Probe with SILAC (Stable Isotope Labeling with Amino acids in Cell culture)-labeled cell proteins to first form a protein-Probe complex, and then add Biotin-labeled azide. Through the CuAAC reaction (Click reaction), a protein-Probe-biotin product is generated.

[0025] (3) Capture the protein-Probe-biotin product through Streptavidin-mediated Pull-down technology.

[0026] (4) The protein-Probe-biotin product is detected by mass spectrometry, and with the help of the "mass tag" introduced by SILAC technology, the identification and relative quantification of proteins are achieved, so as to finally screen and discover the specific binding target protein of the Probe.

[0027] 2. Combine population studies, omics sequencing, PudMed retrieval, and additionally through molecular docking, and screen out that STUB1 may be a target of BP-3 after integration.

[0028] Population studies refer to Li Y, Zhu J, Fan J, Cai S, Fan C, Zhong Y, and Sun L (2020). Associations of urinary levels of phenols and parabens with osteoarthritis among US adults in NHANES 2005 - 2014, Ecotoxicol Environ Saf 192, 110293.

[0029] Omics sequencing: Modeling: After applying and exposing BP - 3 (30 mg / KG / D) to the mouse joint cavity for 3 months, take the articular cartilage for omics sequencing. First, grind the tissue, extract the protein, perform DIA data analysis after protein quantification, and then conduct bioinformatics analysis to screen for differential proteins.

[0030] Molecular docking operation steps:

[0031] (1) Prepare the protein structure and obtain the protein structure:

[0032] Download the crystal structure (PDB format) of the target protein (STUB1) from the PDB database (https: / / www.rcsb.org), and preferably select the structure with high resolution and ligand.

[0033] (2) Prepare the small molecule ligand and obtain the small molecule structure: Download the 3D structure (SDF / MOL2 format) of the bp - 3 small molecule from PubChem (https: / / pubchem.ncbi.nlm.nih.gov).

[0034] (3) Define the docking region (binding site) manually: Use PyMOL or AutoDock Tools to visualize the protein.

[0035] The results show that BP - 3 can dock with the STUB1 molecule, and the docking result is - 7.8 kcal / mol, proving the strong binding of Bp - 3 to STUB1 (see appendix Figure 1 ).

[0036] BP-3-induced damage to articular chondrocytes: In this experiment, human normal chondrocytes C28 / I2 were used. Undifferentiated cells were cultured in cell culture dishes containing 89% high-glucose DMEM (Dulbecco's modified Eagle's medium), 10% FBS (fetal bovine serum), and 1% double antibody (penicillin-streptomycin mixture) in a constant temperature incubator at 37°C and 5% CO2. After treatment with different concentrations of BP-3 (0, 25, 50, 75, 100 μM) with DMSO as the solvent for 48 h, the expression level of STUB1 was detected by quantitative PCR and Western Blot. Among them, the quantitative PCR primers included SEQ ID NO.1 - SEQ ID NO.4.

[0037] STUB1_F1: AAGGAGCAGGGCAATCGTC, SEQ ID NO.1,

[0038] STUB1_R1: AGCGGGTTCCGGGTGAT, SEQ ID NO.2;

[0039] GAPDH_F: TGCACCACCAACTGCTTAG, SEQ ID NO.3,

[0040] GAPDP_R: GATGCAGGGATGATGTTC, SEQ ID NO.4.

[0041] The results proved that BP-3 could induce an increase in STUB1 expression (see attached Figure 2 、 3 ) and showed osteoarthritis-like changes (the index MMP13 reflecting osteoarthritis injury showed an upward trend, while human collagen type II CollagenⅡ, which maintains joint health, showed a downward trend).

[0042] 4. Construction of STUB1 gene knockout vector and verification of the binding of BP-3 to STUB1

[0043] (1) Shanghai Sangon Biotech Co., Ltd. was ordered to knock out the siRNA of the target gene STUB1, and the decrease in the expression level of the target gene mRNA was detected by qPCR (primers: hSTUB1-69-s-CGAGCGCGCAGGAGCUCAA / dT / / dT / , / hSTUB1-69-a: UUGAGCUCCUGCGCGCUCG / dT / / dT / ). The effective decrease of the target protein was verified by protein level detection (Westernblot).

[0044] (2) Transfect the siRNA of the target gene into C28 / I2 chondrocytes. Divide the experimental group into BP-3 exposure group, DMSO solvent control group (CTRL), SiNC transfection reagent control group, and SiSTUB1+BP-3 transfection control group.

[0045] Plate the cells the day before, seed the cells in a culture plate so that the cell confluence reaches 70-80% on the day of transfection. Change to antibiotic-free medium: Replace with fresh antibiotic-free medium 1-2 hours before transfection to reduce the impact of antibiotics on cells and transfection reagents.

[0046] Prepare the siRNA dilution: Dilute the siRNA in serum-free medium (Opti-MEM) (the final concentration is generally 5–50 nM and can be optimized).

[0047] Prepare the transfection reagent dilution: Dilute the transfection reagent in serum-free medium (according to the ratio in the instruction manual).

[0048] Mix and incubate: Mix the above siRNA dilution and transfection reagent dilution, and incubate at room temperature for 15 min to form the siRNA-transfection reagent complex.

[0049] Drop the siRNA-transfection reagent complex directly into the cell culture medium and gently shake to mix evenly. After incubating for 6–8 hours, the medium can be changed to complete medium containing serum and continue the culture. Perform Western blot experiment after a total culture time of 48 hours.

[0050] (3) Results: It can be seen that compared with the DMSO solvent control group, the osteoarthritis injury index MMP13 increased in the BP-3 group, while human collagen type 2 (CollagenⅡ), which maintains joint health, decreased; compared with the BP-3 group, MMP13 decreased and CollagenⅡ increased in the SiSTUB1+BP-3 group, indicating that knocking out STUB1 helps to alleviate chondrocyte injury, suggesting that STUB1 interacts with BP-3 and has an impact on the injury of osteoarthritis, and further indicating that STUB1 protein acts as a BP-3 target in osteoarthritis (see appendix Figure 4 ).

[0051] In this specification, each embodiment is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. For the same or similar parts among the embodiments, reference can be made to each other.

[0052] The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of STUB1 protein as a BP-3 target in osteoarthritis.

2. The use according to claim 1, characterized in that: Regulating the expression of STUB1 protein is used to screen diagnostic reagents for osteoarthritis or screen drugs for alleviating osteoarthritis.

3. Application of reagents for detecting STUB1 gene expression in the preparation of osteoarthritis diagnostic products.

4. The use according to claim 3, characterized in that: The reagent for detecting the expression amount of the STUB1 gene is a kit, and the kit includes a primer set such as SEQ ID NO.1-SEQ ID NO.

4.

5. Application of reagents for detecting STUB1 protein expression in the preparation of osteoarthritis diagnostic products.

6. The use according to claim 3, characterized in that: The reagent for detecting the expression amount of STUB1 protein is a kit, and the kit includes a STUB1 protein standard.

7. Application of STUB1 protein inhibitors in the preparation of drugs for alleviating osteoarthritis caused by BP-3.

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