ABSTRACT
Objective To explore the effect and mechanism of Bajitianwan on preventing D-galactose (D-gal)-induced osteoblast bone loss. Methods Osteoblasts isolated from 24 h old Wistar rats were injured by D-gal and intervened with Bajitianwan extract. The osteoblastic proliferation and differentiation were determined by MTT and alkaline phosphatase (ALP), respectively. The cell reactive oxygen species (ROS) levels were detected by DCFH-DA fluorescent probes. The expression of cellular oxidation-related protein nuclear factor erythroid 2-related factor 2 (Nrf2), phosphorylated protein kinase B (p-AKT), protein kinase B (AKT), heme oxygenase-1 (HO-1), and NADPH quinone oxidoreductase 1 (NQO1) were detected by Western blotting. The intranuclear expression of Nrf2 protein was measured by immunofluorescence. Results Bajitianwan extract had significantly increased the osteoblastic proliferation and differentiation, and significantly reduced the intracellular ROS level. Bajitianwan extract had activated the PI3K/AKT pathway via activating the phosphorylation of AKT in osteoblasts, and promoted NQO1 and HO-1 expression. In addition, Bajitianwan had significantly promoted the expression of Nrf2 in the nucleus of osteoblasts, activating Nrf2 signaling pathway, and further promoted bone formation. Conclusion This study confirmed that Bajitianwan could prevent D-gal injured osteoblastic bone loss for the first time. The mechanism might be related to the regulation of oxidative stress associated PI3K/AKT and Nrf2 signaling pathway.
ABSTRACT
Bearing the largest single-stranded RNA genome in nature, SARS-CoV-2 utilizes sophisticated replication/transcription complexes (RTCs), mainly composed of a network of nonstructural proteins and nucleocapsid protein, to establish efficient infection. Here, we developed an innovative interaction screening strategy based on phase separation in cellulo, namely compartmentalization of protein-protein interactions in cells (CoPIC). Utilizing CoPIC screening, we mapped the interaction network among RTC-related viral proteins. We identified a total of 47 binary interactions among 14 proteins governing replication, discontinuous transcription, and translation of coronaviruses. Further exploration via CoPIC led to the discovery of extensive ternary complexes composed of these components, which infer potential higher-order complexes. Taken together, our results present an efficient, and robust interaction screening strategy, and indicate the existence of a complex interaction network among RTC-related factors, thus opening up new opportunities to understand SARS-CoV-2 biology and develop therapeutic interventions for COVID-19.
ABSTRACT
The ongoing coronavirus disease 2019 (COVID-19) pandemic has raised an urgent need to develop effective therapeutics against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As a potential antiviral drug target, the nucleocapsid (N) protein of SARS-CoV-2 functions as a viral RNA chaperone and plays vital and multifunctional roles during the life cycle of coronavirus1-3. In this study, we discovered that the N protein of SARS-CoV-2 undergoes liquid-liquid phase separation (LLPS) both in vitro and in vivo, which is further modulated by viral RNA. In addition, we found that, the core component of the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, nsp12, preferentially partitions into the N protein condensates. Moreover, we revealed that, two small molecules, i.e., CVL218 and PJ34, can be used to intervene the N protein driven phase separation and loosen the compact structures of the condensates of the N-RNA-nsp12 complex of SARS-CoV-2. The discovery of the LLPS-mediated interplay between N protein and nsp12 and the corresponding modulating compounds illuminates a feasible way to improve the accessibility of antiviral drugs (e.g., remdesivir) to their targets (e.g., nsp12/RdRp), and thus may provide useful hints for further development of effective therapeutic strategies against SARS-CoV-2.
