ABSTRACT
Pulmonary fibrosis (PF) is characterized by myofibroblast activation, which can be triggered by oxidative stress. In this study, we investigated the antifibrotic effect of the ethyl acetate extract of Salvia miltiorrhiza (EASM) on PF and examined the underlying molecular mechanism. EASM suppressed myofibroblast activation with reduced extracellular matrix deposition in the lungs of mice subjected to bleomycin (BLM) challenge, demonstrating the inhibitory effects on PF. EASM positively alleviated oxidative stress by upregulating nuclear factor-erythroid 2-related factor 2 (Nrf2) and concomitantly downregulating NADPH oxidase 4 (Nox4) in the lungs of BLM-treated mice. This effect was also observed in an in vitro model of transforming growth factor beta 1 (TGF-ß1)-stimulated fibroblast activation. EASM reduced reactive oxygen species (ROS) generation in fibroblasts by stabilizing Nrf2 protein with promoting kelch-like ECH-associated protein 1 (Keap1) degradation. Nrf2 knockdown in the lungs of BLM-treated mice diminished the inhibitory effects of EASM on fibrosis, providing evidence in vivo to address the unique role of Nrf2. Additionally, EASM inhibited TGF-ß1/Smad3 signaling by downregulating protein kinase C delta (PKC-δ) and Smad3 phosphorylation (p-Smad3), which led to suppression of the TGF-ß1-induced fibrogenic response. These results indicate that EASM exhibits potent antifibrotic activity in vitro and in vivo, which might be associated with activation of Nrf2 pathway and inhibition of TGF-ß1/Smad3 pathway. Our findings support that EASM may act as an effective antifibrotic remedy for PF.
Subject(s)
Drugs, Chinese Herbal/administration & dosage , NADPH Oxidase 4/metabolism , NF-E2-Related Factor 2/metabolism , Pulmonary Fibrosis/drug therapy , Reactive Oxygen Species/metabolism , Salvia miltiorrhiza/chemistry , Animals , Female , Humans , Kelch-Like ECH-Associated Protein 1/genetics , Kelch-Like ECH-Associated Protein 1/metabolism , Lung/drug effects , Lung/metabolism , Mice , Mice, Inbred C57BL , Myofibroblasts/drug effects , Myofibroblasts/metabolism , NADPH Oxidase 4/genetics , NF-E2-Related Factor 2/genetics , Pulmonary Fibrosis/genetics , Pulmonary Fibrosis/metabolism , Smad3 Protein/metabolism , Transforming Growth Factor beta/metabolismABSTRACT
AIMS: Pulmonary fibrosis (PF) is characterized by myofibroblast activation through oxidative stress. However, the precise regulation of myofibroblast transdifferentiation remains largely uncharacterized. RESULTS: In this study, we found that tanshinone IIA (Tan-IIA), an active component in the root of Salvia miltiorrhiza Bunge, can suppress reactive oxygen species (ROS)-mediated activation of myofibroblast and reduce extracellular matrix deposition in bleomycin (BLM)-challenged mice through the regulation of nuclear factor-erythroid 2-related factor 2 (Nrf2). Additionally, Tan-IIA restored redox homeostasis by upregulating Nrf2 with NADPH oxidase 4 suppression and effectively prevented myofibroblast activation by blocking ROS-mediated protein kinase C delta (PKCδ)/Smad3 signaling. Nrf2 knockdown in the fibroblasts and the lungs of BLM-treated mice reduced the inhibitory effects of Tan-IIA, indicating the essential role of Nrf2 in the Tan-IIA activity. Tan-IIA impaired the binding of kelch-like ECH-associated protein 1 (Keap1) to Nrf2 by promoting the degradation of Keap1 and thereby increasing Nrf2 induction by protecting Nrf2 stability against ubiquitination and proteasomal degradation. Importantly, we also found that the glutamate anaplerotic pathway was involved in energy generation and biosynthesis in activated myofibroblasts and their proliferation. Tan-IIA shunted glutaminolysis into glutathione (GSH) production by activating Nrf2, resulting in the reduction of glutamate availability for tricarboxylic acid cycle. Ultimately, myofibroblast activation was prevented by impairing cell proliferation. Innovation and Conclusion: In addition to the regulation of redox homeostasis, our work showed that Tan-IIA activated Nrf2/GSH signaling pathway to limit glutaminolysis in myofibroblast proliferation, which provided further insight into the critical function of Nrf2 in PF.
Subject(s)
Abietanes/pharmacology , Glutamine/metabolism , Homeostasis , NF-E2-Related Factor 2/agonists , Oxidation-Reduction , Pulmonary Fibrosis/etiology , Pulmonary Fibrosis/metabolism , Animals , Cell Transdifferentiation , Female , Fibroblasts/metabolism , Gene Expression , Glutathione/metabolism , Humans , Immunohistochemistry , Mice , Models, Biological , Myofibroblasts/metabolism , NADPH Oxidase 4/genetics , NADPH Oxidase 4/metabolism , NF-E2-Related Factor 2/chemistry , NF-E2-Related Factor 2/metabolism , Pulmonary Fibrosis/drug therapy , Pulmonary Fibrosis/pathology , Reactive Oxygen Species/metabolismABSTRACT
The osteogenic differentiation of mesenchymal stem cells (MSCs) is governed by multiple mechanisms. Growing evidence indicates that ubiquitin-dependent protein degradation is critical for the differentiation of MSCs and bone formation; however, the function of ubiquitin-specific proteases, the largest subfamily of deubiquitylases, remains unclear. Here, we identify USP34 as a previously unknown regulator of osteogenesis. The expression of USP34 in human MSCs increases after osteogenic induction while depletion of USP34 inhibits osteogenic differentiation. Conditional knockout of Usp34 from MSCs or pre-osteoblasts leads to low bone mass in mice. Deletion of Usp34 also blunts BMP2-induced responses and impairs bone regeneration. Mechanically, we demonstrate that USP34 stabilizes both Smad1 and RUNX2 and that depletion of Smurf1 restores the osteogenic potential of Usp34-deficient MSCs in vitro Taken together, our data indicate that USP34 is required for osteogenic differentiation and bone formation.
Subject(s)
Bone Morphogenetic Protein 2/metabolism , Cell Differentiation , Mesenchymal Stem Cells/metabolism , Osteogenesis , Signal Transduction , Ubiquitin-Specific Proteases/metabolism , Animals , Bone Morphogenetic Protein 2/genetics , Bone Regeneration/genetics , Gene Knockdown Techniques , Humans , Mesenchymal Stem Cells/cytology , Mice , Mice, Knockout , Osteoblasts/cytology , Osteoblasts/metabolism , Ubiquitin-Specific Proteases/geneticsABSTRACT
OBJECTIVE: To develop a new local delivery system, zoledronic-acid-loaded chitosan/calcium phosphate ceramic, and to determine its characterization and in vitro response of osteoblast cells. METHODS: Zoledronic-acid-loaded chitosan/calcium phosphate ceramic were prepared by solution casting method at a concentration of 10(-5), 10(-4), and 10(-3) mol/L, respectively. The physicochemical properties of the resulting materials were determined using SEM and FTIR. Drug absorbance was measured using CCK-8 colorimetric assay and alkaline phosphatase assay to detect the effect of drug-loaded materials on the proliferation and differentiation of osteoblasts. RESULTS: After ZOL loading, SEM showed that porous calcium phosphate ceramic was coated with chitosan evenly. The IR spectra indicated that drug absorption peaks were shifted and a new one was formed for the drug-loaded biomaterials. The material at the highest concentration could inhibit the proliferation and alkaline phosphatase activities of osteoblast cells, but no such effect was found at a drug-loading concentration of 10(-4)-10(-5) mol/L. CONCLUSION: We confirmed that the local delivery system in this study has ability of loading ZOL. The biomaterial with high drug concentrations inhibits the proliferation and differentiation of osteoblasts, but not when the drug concentrations are low.
