Resveratrol counteracts bone loss via mitofilin-mediated osteogenic improvement of mesenchymal stem cells in senescence-accelerated mice
Yajie LvYi YangBing‐Dong SuiCheng‐Hu HuPan ZhaoLi LiaoJihua ChenLiqiang ZhangTong-Tao YangShaofeng ZhangYan Jin
91
Citation
134
Reference
10
Related Paper
Citation Trend
Abstract:
Rational: Senescence of mesenchymal stem cells (MSCs) and the related functional decline of osteogenesis have emerged as the critical pathogenesis of osteoporosis in aging.Resveratrol (RESV), a small molecular compound that safely mimics the effects of dietary restriction, has been well documented to extend lifespan in lower organisms and improve health in aging rodents.However, whether RESV promotes function of senescent stem cells in alleviating age-related phenotypes remains largely unknown.Here, we intend to investigate whether RESV counteracts senescence-associated bone loss via osteogenic improvement of MSCs and the underlying mechanism.Methods: MSCs derived from bone marrow (BMMSCs) and the bone-specific, senescence-accelerated, osteoblastogenesis/osteogenesis-defective mice (the SAMP6 strain) were used as experimental models.In vivo application of RESV was performed at 100 mg/kg intraperitoneally once every other day for 2 months, and in vitro application of RESV was performed at 10 μM.Bone mass, bone formation rates and osteogenic differentiation of BMMSCs were primarily evaluated.Metabolic statuses of BMMSCs and the mitochondrial activity, transcription and morphology were also examined.Mitofilin expression was assessed at both mRNA and protein levels, and short hairpin RNA (shRNA)-based gene knockdown was applied for mechanistic experiments.Results: Chronic intermittent application of RESV enhances bone formation and counteracts accelerated bone loss, with RESV improving osteogenic differentiation of senescent BMMSCs.Furthermore, in rescuing osteogenic decline under BMMSC senescence, RESV restores cellular metabolism through mitochondrial functional recovery via facilitating mitochondrial autonomous gene transcription.Molecularly, in alleviating senescence-associated mitochondrial disorders of BMMSCs, particularly the mitochondrial morphological alterations, RESV upregulates Mitofilin, also known as inner membrane protein of mitochondria (Immt) or Mic60, which is the core component of the mitochondrial contact site and cristae organizing system (MICOS).Moreover, Mitofilin is revealed to be indispensable for mitochondrial homeostasis and osteogenesis of BMMSCs, and that insufficiency of Mitofilin leads to BMMSC senescence and bone loss.More importantly, Mitofilin mediates resveratrol-induced mitochondrial and osteogenic improvements of BMMSCs in senescence.Conclusion: Our findings uncover osteogenic functional improvements of senescent MSCs as critical impacts in anti-osteoporotic practice of RESV, and unravel Mitofilin as a novel mechanism mediating RESV promotion on mitochondrial function in stem cell senescence.Keywords:
Senescence
Cite
Citations (12)
Cite
Citations (4)
Gene knockout
Cite
Citations (3)
Rapidly activating and inactivating A-type K+ currents (IA) encoded by Kv4.2 and Kv4.3 pore-forming (α) subunits of the Kv4 subfamily are key regulators of neuronal excitability. Previous studies have suggested a role for Kv4.1 α-subunits in regulating the firing properties of mouse suprachiasmatic nucleus (SCN) neurons. To test this, we utilized an RNA-interference strategy to knockdown Kv4.1, acutely and selectively, in the SCN. Current-clamp recordings revealed that the in vivo knockdown of Kv4.1 significantly (p < 0.0001) increased mean ± SEM repetitive firing rates in SCN neurons during the day (6.4 ± 0.5 Hz) and at night (4.3 ± 0.6 Hz), compared with nontargeted shRNA-expressing SCN neurons (day: 3.1 ± 0.5 Hz; night: 1.6 ± 0.3 Hz). IA was also significantly (p < 0.05) reduced in Kv4.1-targeted shRNA-expressing SCN neurons (day: 80.3 ± 11.8 pA/pF; night: 55.3 ± 7.7 pA/pF), compared with nontargeted shRNA-expressing (day: 121.7 ± 10.2 pA/pF; night: 120.6 ± 16.5 pA/pF) SCN neurons. The magnitude of the effect of Kv4.1-targeted shRNA expression on firing rates and IA was larger at night. In addition, Kv4.1-targeted shRNA expression significantly (p < 0.001) increased mean ± SEM nighttime input resistance (Rin; 2256 ± 166 MΩ), compared to nontargeted shRNA-expressing SCN neurons (1143 ± 93 MΩ). Additional experiments revealed that acute knockdown of Kv4.1 significantly (p < 0.01) shortened, by ∼0.5 h, the circadian period of spontaneous electrical activity, clock gene expression and locomotor activity demonstrating a physiological role for Kv4.1-encoded IA channels in regulating circadian rhythms in neuronal excitability and behavior.
Cite
Citations (27)
Doublecortin
Gene knockout
Knockout mouse
Dicer
Cite
Citations (91)
Cite
Citations (85)
Objective: To establish the gene knockdown system (RNAi) using different Multiple Myeloma Cell lines (MMCL). Material and Methods: RNAi is generally used in biomedical research to study the outcome of knockdown of gene expression. In this research, retroviral-shRNA expression constructs based on micro RNA-adapted short hairpin RNA (sh RNA mir) were designed and constructed. The sequence of the cloned fragments in the vector constructs was determined, and the effect of constructs in MMCLs was examined using qRT-PCR and Western blot analysis. Results: In this work, we established a method for making retroviral-shRNA expression constructs based on micro RNA-adapted short hairpin RNA (sh RNA mir) design. The HGF producing human myeloma cell line JJN-3 transduced with lentiviral HGF-shRNA transduction particles were analyzed for their effect on the HGF gene expression. HGF knockdown MMCL was analyzed both at mRNA level and protein levels. Two out of five clones of myeloma cell line JJN-3 (JJN3-sh3308 and JJN3-sh47137) were transduced with lentiviral HGF–shRNA transduction particles were showing 50-60 % knockdown in their HGF mRNA and protein expression. Conclusion: HGF knockdown cell lines can be used as tissue culture models to investigate the role of HGF protein in the pathology of multiple myeloma. Further, the retroviral-HGF-shRNA expression constructs can study HGF response in various multiple myeloma via gene knockdown studies.
Cite
Citations (0)
Trans-acting siRNA
Cite
Citations (0)
Short-hairpin RNA (shRNA)-mediated gene knockdown is a powerful tool for targeted gene silencing and an emerging novel therapeutic strategy. Recent publications, however, reported unexpected toxicity after utilizing viral-mediated shRNA knockdown in vivo. Thus, it is currently unclear whether shRNA-mediated knockdown strategy can be used as a safe and efficient tool for gene silencing. In this study, we have generated rAAV vectors expressing shRNAs targeting the rat tyrosine hydroxylase (TH) mRNA (shTH) for testing the efficacy of in vivo TH knockdown in the nigral dopaminergic neurons. At high titers, not only the shTH vectors but also the scrambled and green fluorescence protein (GFP)-only controls caused cell death. In a dose-response study, we identified a dose window leading to >60% decrease in TH(+) neurons without any change in vesicular monoamine transporter-2 (VMAT2) expression. Moreover, using the safe and efficient dose, we showed that dopamine (DA) synthesis rate was significantly reduced and this lead to emergence of motor deficits in the shTH-expressing rats. Interestingly, these animals showed very robust and long-lasting recovery after a single systemic L-3,4-dihydroxyphenylalanine (L-DOPA) administration beyond what can be achieved in 6-hydroxydopamine (6-OHDA)-lesioned rats. Our results have implications for both mechanistic and therapeutic studies utilizing long-term shRNA-mediated gene silencing in the nigrostriatal projection system.
Cite
Citations (71)
The regenerative and immunomodulatory properties of mesenchymal stem cells (MSCs) have laid a sound foundation for their clinical application in various diseases. However, the clinical efficiency of MSC treatments varies depending on certain cell characteristics. Among these, the roles of cell aging or senescence cannot be excluded. Despite their stemness, evidence of senescence in MSCs has recently gained attention. Many factors may contribute to the senescence of MSCs, including MSC origin (biological niche), donor conditions (age, obesity, diseases, or unknown factors), and culture conditions in vitro. With the rapidly increasing prevalence of diabetes mellitus (DM) and gestational diabetes mellitus (GDM), the effects of hyperglycemia on the senescence of MSCs should be evaluated to improve the application of autologous MSCs. This review aims to present the available data on the senescence of MSCs, its relationship with hyperglycemia, and the strategies to suppress the senescence of MSCs in a hyperglycemic environment.
Senescence
Regenerative Medicine
Cite
Citations (32)