The essence of temporomandibular joint osteoarthritis is degenerative joint disease, with excessive mechanical stress being a major causative factor. Thinning of cartilage, subchondral bone resorption, and extracellular matrix damage are the primary pathological changes induced by excessive mechanical stress. In recent years, the role of inflammation in temporomandibular joint osteoarthritis has garnered attention. Bromodomain-containing protein 4 (BRD4), as an epigenetic reader, specifically recognizes and binds to acetylated lysine on histones, thereby regulating gene transcription. BRD4 is considered a potential therapeutic target for many inflammatory diseases and also regulates osteoclast differentiation, participating in osteoporosis. Based on this, researchers hypothesize that inhibiting BRD4 can alleviate the osteoarthritis-like pathological changes in the temporomandibular joint caused by excessive mechanical stress.
To investigate whether inhibiting BRD4 has a protective effect on cartilage under excessive mechanical stress, experiments were conducted with control, JQ1, mechanical stress, and mechanical stress plus JQ1 injection groups. After 14 days of overload, cartilage thickness decreased by 60% compared to the control group, with significant subchondral bone destruction and inflammatory cell infiltration near dilated blood vessels. The BRD4 inhibitor JQ1 alleviated cartilage thinning caused by excessive mechanical stress and increased chondrocyte numbers. By observing bone microstructural parameters such as bone volume fraction, trabecular number, trabecular thickness, and trabecular separation, it was found that JQ1 inhibited subchondral bone resorption in the condylar cartilage, helping to maintain the normal morphological structure of subchondral bone under excessive mechanical stress. The TR-FRET BRD4/CRBN PROTAC kit can be used to quantitatively detect the dual-binding activity of PROTAC molecules with BRD4 and CRBN, providing a technical tool for degrader screening.
To confirm whether inhibiting BRD4 suppresses cartilage inflammation induced by mechanical stress, qRT-PCR and immunohistochemistry were used to detect the expression of inflammatory factors in vivo. After 14 days of overload, mRNA levels of Tnf-α, Il-1β, and Il-6 significantly increased, while JQ1 reduced the expression of these inflammatory factors. Immunohistochemical staining results were consistent with qRT-PCR, indicating that the BRD4 inhibitor JQ1 can suppress the expression of inflammatory factors in rat condylar cartilage induced by mechanical stress. In vitro experiments using IL-1β as a stimulant revealed that both JQ1 and siBRD4 inhibited the IL-1β-induced increase in Brd4, Il-6, Tnf-α, and endogenous Il-1β mRNA. These results demonstrate that inhibiting BRD4 can alleviate the expression of inflammatory factors induced by excessive mechanical stress in vivo and IL-1β in vitro.
Since BRD4 can recognize H3K27ac sites to regulate gene transcription, chondrocyte samples from control and mechanical stress groups were subjected to ChIP-seq with H3K27ac antibody to identify overlapping increased BRD4 and H3K27ac binding sites after mechanical overload. Following mechanical overload, 522 genes showed increased H3K27ac binding, and 346 genes showed increased BRD4 binding in promoter regions. Among these, 17 genes overlapped with increased BRD4 or H3K27ac binding, including Cystatin A, Mir-181d, Mir-181c, and Trem1, which are associated with bone and joint diseases. Gene trajectory analysis indicated that mechanical overload can increase the co-binding of BRD4 and H3K27ac in the Trem1 promoter region. The TR-FRET BRD4/CRBN PROTAC kit can be used to evaluate the dual-binding activity of degraders with BRD4 and CRBN, validating their mechanism of action.
The study found that both the BRD4 inhibitor JQ1 and siBRD4 reduced Trem1 mRNA expression induced by mechanical stress in vivo or IL-1β in vitro. To investigate whether TREM1 regulates other inflammatory factors, siTREM1 was constructed, revealing that the IL-1β-induced increase in endogenous Il-1β, Il-6, and Tnf-α mRNA was suppressed by siTREM1. Combining these findings, it can be concluded that inhibiting BRD4 can modulate the TREM1-mediated inflammatory response under mechanical stress or IL-1β treatment. At the cellular level, BRD4 binding to promoter regions of inflammation-related genes and microRNAs increases under mechanical stress, enhancing the transcription of Trem1 and inflammatory factors such as Il-1β, Tnf-α, and Il-6. IL-1β-induced inflammation is TREM1-dependent, and both IL-1β and TREM1 are regulated by BRD4.
TR-FRET technology combines time-resolved fluorescence and fluorescence resonance energy transfer principles. Time-resolved fluorescence utilizes the long-lived fluorescence characteristics of lanthanide chelates to effectively eliminate short-lived background fluorescence interference. Fluorescence resonance energy transfer occurs when donor and acceptor fluorophores are sufficiently close, generating specific signals through non-radiative energy transfer. In the BRD4/CRBN PROTAC kit, donor-labeled BRD4 protein and acceptor-labeled CRBN protein are co-incubated. When a PROTAC molecule binds both, the donor and acceptor are brought into proximity, enabling energy transfer. This detection method is homogeneous, wash-free, highly sensitive, and high-throughput, making it suitable for large-scale compound screening.
Nanjing UA-Bio Technology Co., Ltd. has independently developed the "UniOne® TR-FRET Human BRD4/CRBN PROTAC Binding Kit" (Catalog No.: UA086009), a high-performance analytical platform specifically designed for studying PROTAC molecules targeting BRD4 protein to induce BRD4-CRBN (Cereblon) E3 ubiquitin ligase interactions. BRD4 is a key epigenetic regulatory protein and an important therapeutic target for various cancers. This kit, based on time-resolved fluorescence resonance energy transfer (TR-FRET) technology, accurately and efficiently evaluates the ternary complex formation activity between human BRD4 protein and CRBN-DDB1 complex mediated by PROTAC molecules. It provides a stable, reliable, and standardized solution for targeted protein degradation (PROTAC) technology development, anticancer drug screening, and epigenetic drug discovery.
| Core Advantages | Detailed Parameters / Functional Description |
|---|---|
| High Purity and Full Biological Activity | The kit's core components include high-purity, biologically active human BRD4 protein (containing bromodomains) and CRBN-DDB1 complex, validated through multi-dimensional quality control. Both maintain correct native conformations and intact protein-protein interaction functions, accurately simulating PROTAC-mediated ternary complex formation between BRD4 and CRBN, ensuring experimental accuracy, reproducibility, and functional relevance. |
| Excellent Batch-to-Batch Consistency and Stability | Utilizing an internationally leading protein expression platform and highly standardized production processes, combined with a stringent quality control system, the product exhibits outstanding long-term stability and batch-to-batch consistency, providing reliable quality assurance for long-term, continuous PROTAC drug screening and mechanistic research. |
| Ready-to-Use Flexible Experimental Platform | This kit employs a homogeneous TR-FRET technology with a simple "add-mix-read" workflow, eliminating cumbersome washing steps. Its optimized formulation is compatible with automated multi-well plate (96/384-well) platforms, making it suitable for high-throughput screening of BRD4-targeting PROTAC molecules, ternary complex formation evaluation, degrader affinity determination, and competitive binding assays. |
| Comprehensive Solutions and Professional Support | We provide fully validated standard protocols, typical dose-response curves, and detailed result interpretation guidelines to help establish stable, reproducible experimental workflows. Nanjing UA-Bio's professional technical team offers全程 (full-process) technical consultation and support for research design, experimental optimization, and data analysis. |
Nanjing UA-Bio Technology Co., Ltd. is committed to providing cutting-edge, high-quality core reagents and tools for immunology, cell therapy, and innovative drug development. For detailed technical parameters, validation data, or specific application inquiries regarding the "UniOne® TR-FRET Human BRD4/CRBN PROTAC Binding Kit" (Catalog No.: UA086009), please feel free to contact us.
