Yusuke Kubo, Anna Theresa Tangarife Bodensiek, Sebastian Lippross, Marek Weiler, Nina Reinhardt, Matias de la Fuente Klein, Matthias Gatz, Ulf Krister Hofmann, Fabian Kießling, Vera Jankowski, Jaana-Sophia Kern, Marta Rizk, Elizabeth Rosado Balmayor, Anna Bock, Michael Wolf, Thomas Pufe, Mersedeh Tohidnezhad
{"title":"低强度脉冲超声（LIPUS）诱导的球形培养中Nrf2/ARE和NFκB应答与成骨细胞功能的可能关联","authors":"Yusuke Kubo, Anna Theresa Tangarife Bodensiek, Sebastian Lippross, Marek Weiler, Nina Reinhardt, Matias de la Fuente Klein, Matthias Gatz, Ulf Krister Hofmann, Fabian Kießling, Vera Jankowski, Jaana-Sophia Kern, Marta Rizk, Elizabeth Rosado Balmayor, Anna Bock, Michael Wolf, Thomas Pufe, Mersedeh Tohidnezhad","doi":"10.1016/j.ultrasmedbio.2025.03.013","DOIUrl":null,"url":null,"abstract":"<p><strong>Objective: </strong>NFκB negatively affects bone metabolism through inflammatory pathways, whereas Nrf2 benefits it by regulating antioxidant/detoxifying enzymes through antioxidant response element (ARE) activity. We investigated whether low-intensity pulsed ultrasound (LIPUS) enhances osteogenic differentiation in 3D culture and whether it affects osteoblastic differentiation-associated markers such as calcium and alkaline phosphatase (ALP), and the regulation of NFκB and/or ARE.</p><p><strong>Methods: </strong>Murine MC3T3-E1 preosteoblasts were used to produce 3D-spheroids, which were treated with LIPUS. Time-dependent morphological change of spheroids was evaluated by microscopy, histology, and micro-CT. To analyze changes in ARE levels, a combination of LIPUS with an optimal concentration and timing of the Nrf2-inducer sulforaphane was applied. Osteoblast differentiation and mineralization were achieved through stimulation with differentiation medium for 21-28 days, during which LIPUS was used every day at 24-hour intervals. Differentiation was evaluated using calcium and ALP assay. Finally, cells were transduced with SIN-lenti-NFκB/SIN-lenti-ARE construct, and the effects of single-shot LIPUS on NFκB and ARE were evaluated using Nano-Glo® Luciferase Assay.</p><p><strong>Results: </strong>Morphologically, in LIPUS group, shrinkage behaviour, which may correlate with cell differentiation, was observed to be more pronounced. NFκB activity with LIPUS was significantly lower than without treatment, whereas ARE activity showed significant increase only immediately after LIPUS treatment. Conversely, LIPUS enhanced ARE activity in combination with sulforaphane administration. After 28 days, the calcium content and ALP activity of LIPUS-treated spheroids increased significantly.</p><p><strong>Conclusion: </strong>This study suggests that LIPUS may have NFκB-downregulating, limited ARE-raising effects in combination with sulforaphane, and elevating osteoblast mineralization three-dimensionally. Thus, LIPUS could be useful for treating fractures in conditions of oxidative stress and hyper-inflammation.</p>","PeriodicalId":49399,"journal":{"name":"Ultrasound in Medicine and Biology","volume":" ","pages":""},"PeriodicalIF":2.4000,"publicationDate":"2025-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Possible Association of Nrf2/ARE and NFκB Response to Osteoblast Function in Spheroid Culture Induced by Low-Intensity Pulsed Ultrasound (LIPUS).\",\"authors\":\"Yusuke Kubo, Anna Theresa Tangarife Bodensiek, Sebastian Lippross, Marek Weiler, Nina Reinhardt, Matias de la Fuente Klein, Matthias Gatz, Ulf Krister Hofmann, Fabian Kießling, Vera Jankowski, Jaana-Sophia Kern, Marta Rizk, Elizabeth Rosado Balmayor, Anna Bock, Michael Wolf, Thomas Pufe, Mersedeh Tohidnezhad\",\"doi\":\"10.1016/j.ultrasmedbio.2025.03.013\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Objective: </strong>NFκB negatively affects bone metabolism through inflammatory pathways, whereas Nrf2 benefits it by regulating antioxidant/detoxifying enzymes through antioxidant response element (ARE) activity. We investigated whether low-intensity pulsed ultrasound (LIPUS) enhances osteogenic differentiation in 3D culture and whether it affects osteoblastic differentiation-associated markers such as calcium and alkaline phosphatase (ALP), and the regulation of NFκB and/or ARE.</p><p><strong>Methods: </strong>Murine MC3T3-E1 preosteoblasts were used to produce 3D-spheroids, which were treated with LIPUS. Time-dependent morphological change of spheroids was evaluated by microscopy, histology, and micro-CT. To analyze changes in ARE levels, a combination of LIPUS with an optimal concentration and timing of the Nrf2-inducer sulforaphane was applied. Osteoblast differentiation and mineralization were achieved through stimulation with differentiation medium for 21-28 days, during which LIPUS was used every day at 24-hour intervals. Differentiation was evaluated using calcium and ALP assay. Finally, cells were transduced with SIN-lenti-NFκB/SIN-lenti-ARE construct, and the effects of single-shot LIPUS on NFκB and ARE were evaluated using Nano-Glo® Luciferase Assay.</p><p><strong>Results: </strong>Morphologically, in LIPUS group, shrinkage behaviour, which may correlate with cell differentiation, was observed to be more pronounced. NFκB activity with LIPUS was significantly lower than without treatment, whereas ARE activity showed significant increase only immediately after LIPUS treatment. Conversely, LIPUS enhanced ARE activity in combination with sulforaphane administration. After 28 days, the calcium content and ALP activity of LIPUS-treated spheroids increased significantly.</p><p><strong>Conclusion: </strong>This study suggests that LIPUS may have NFκB-downregulating, limited ARE-raising effects in combination with sulforaphane, and elevating osteoblast mineralization three-dimensionally. Thus, LIPUS could be useful for treating fractures in conditions of oxidative stress and hyper-inflammation.</p>\",\"PeriodicalId\":49399,\"journal\":{\"name\":\"Ultrasound in Medicine and Biology\",\"volume\":\" \",\"pages\":\"\"},\"PeriodicalIF\":2.4000,\"publicationDate\":\"2025-04-28\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Ultrasound in Medicine and Biology\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1016/j.ultrasmedbio.2025.03.013\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"ACOUSTICS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Ultrasound in Medicine and Biology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1016/j.ultrasmedbio.2025.03.013","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"ACOUSTICS","Score":null,"Total":0}
Possible Association of Nrf2/ARE and NFκB Response to Osteoblast Function in Spheroid Culture Induced by Low-Intensity Pulsed Ultrasound (LIPUS).
Objective: NFκB negatively affects bone metabolism through inflammatory pathways, whereas Nrf2 benefits it by regulating antioxidant/detoxifying enzymes through antioxidant response element (ARE) activity. We investigated whether low-intensity pulsed ultrasound (LIPUS) enhances osteogenic differentiation in 3D culture and whether it affects osteoblastic differentiation-associated markers such as calcium and alkaline phosphatase (ALP), and the regulation of NFκB and/or ARE.
Methods: Murine MC3T3-E1 preosteoblasts were used to produce 3D-spheroids, which were treated with LIPUS. Time-dependent morphological change of spheroids was evaluated by microscopy, histology, and micro-CT. To analyze changes in ARE levels, a combination of LIPUS with an optimal concentration and timing of the Nrf2-inducer sulforaphane was applied. Osteoblast differentiation and mineralization were achieved through stimulation with differentiation medium for 21-28 days, during which LIPUS was used every day at 24-hour intervals. Differentiation was evaluated using calcium and ALP assay. Finally, cells were transduced with SIN-lenti-NFκB/SIN-lenti-ARE construct, and the effects of single-shot LIPUS on NFκB and ARE were evaluated using Nano-Glo® Luciferase Assay.
Results: Morphologically, in LIPUS group, shrinkage behaviour, which may correlate with cell differentiation, was observed to be more pronounced. NFκB activity with LIPUS was significantly lower than without treatment, whereas ARE activity showed significant increase only immediately after LIPUS treatment. Conversely, LIPUS enhanced ARE activity in combination with sulforaphane administration. After 28 days, the calcium content and ALP activity of LIPUS-treated spheroids increased significantly.
Conclusion: This study suggests that LIPUS may have NFκB-downregulating, limited ARE-raising effects in combination with sulforaphane, and elevating osteoblast mineralization three-dimensionally. Thus, LIPUS could be useful for treating fractures in conditions of oxidative stress and hyper-inflammation.
期刊介绍：
Ultrasound in Medicine and Biology is the official journal of the World Federation for Ultrasound in Medicine and Biology. The journal publishes original contributions that demonstrate a novel application of an existing ultrasound technology in clinical diagnostic, interventional and therapeutic applications, new and improved clinical techniques, the physics, engineering and technology of ultrasound in medicine and biology, and the interactions between ultrasound and biological systems, including bioeffects. Papers that simply utilize standard diagnostic ultrasound as a measuring tool will be considered out of scope. Extended critical reviews of subjects of contemporary interest in the field are also published, in addition to occasional editorial articles, clinical and technical notes, book reviews, letters to the editor and a calendar of forthcoming meetings. It is the aim of the journal fully to meet the information and publication requirements of the clinicians, scientists, engineers and other professionals who constitute the biomedical ultrasonic community.
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