Resveratrol counteracts bone loss via mitofilin-mediated osteogenic improvement of mesenchymal stem cells in senescence-accelerated mice

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: Mitofilin/IMMT/Mic60; mesenchymal stem cells; osteogenesis; osteoporosis; resveratrol; senescence-accelerated mice.

Figure 1

Resveratrol enhances bone formation and counteracts accelerated bone loss in SAMP6 mice. (A-C) Representative 2D section and 3D reconstruction of micro-CT images (A) and quantitative analysis of trabecular bone volume (B) and bone mineral density (C) in distal femora. Bars: 500 μm (2D images) and 50 μm (3D images). (D-F) Representative images of calcein double labeling (D) with quantification of mineral apposition rates (E) and bone formation rates (F) in distal femora. Bars: 25 μm. 4-month-old SAMR1 and SAMP6 mice were treated with either resveratrol (100 mg/kg i.p., every other day for 2 months) or the DMSO (5%) solvent control. n = 4-5 per group. Data represent mean ± SD. *P < 0.05; NS, not significant (P > 0.05). Data were analyzed using ANOVA followed by Newman-Keuls post-hoc tests.

Figure 2

Resveratrol improves osteogenic differentiation of BMMSCs derived from SAMP6 mice. (A-C) Representative images of ALP staining (A) with quantification of ALP grey value of staining (B) and ALP activity (C) in osteogenic differentiation of BMMSCs. Bars: 5 mm. (D-F) Representative images of alizarin red staining (D) with quantification of mineralized nodules (E) and area (F) in osteogenic differentiation of BMMSCs. Bars: 5 mm. (G, H) qRT-PCR analysis of mRNA expression (G) and western blot analysis of protein expression (H) levels of osteogenic marker genes in osteogenic differentiation of BMMSCs. BMMSCs from 4-month-old SAMP6 mice were treated with either resveratrol (10 μM) or the DMSO (0.001%) solvent control. n = 3 per group. Data represent mean ± SD. *P < 0.05; NS, not significant (P > 0.05). Data were analyzed using ANOVA followed by Newman-Keuls post-hoc tests.

Figure 3

Resveratrol improves mitochondrial functionality and transcription in BMMSCs derived from SAMP6 mice. (A) Quantitative analysis of ATP production versus ADP ratio in BMMSCs. (B, C) DCFDA detection of total ROS level in BMMSCs (B) with flow cytometric quantitative analysis of fluorescence intensity (C). Bars: 10 μm. (D, E) Rhodamine 123 detection of mitochondrial membrane potential of BMMSCs (D) with quantitative analysis of fluorescence intensity (E). Bars: 10 μm. (F) Kinetic analysis of oxygen consumption as an index of mitochondrial OXPHOS activity in cultured BMMSCs. (G, H) qRT-PCR analysis of mRNA expression levels of nuclear-encoded (G) and mtDNA-encoded (H) gene representatives for mitochondrial complex subunits of BMMSCs. BMMSCs from 4-month-old SAMP6 mice were treated with either resveratrol (10 μM) or the DMSO (0.001%) solvent control. n = 3 per group. Data represent mean ± SD. *P < 0.05; NS, not significant (P > 0.05). Data were analyzed using ANOVA followed by Newman-Keuls post-hoc tests.

Figure 4

Resveratrol promotes Mitofilin expression and restores mitochondrial morphology in BMMSCs from SAMP6 mice. (A-D) Representative transmission electron microscopy images (A) and quantitative analysis of mitochondrial number (B) and average area (C) per cell as well as the ratio of mitochondrial inner membrane versus outer membrane (D) in BMMSCs. Bars: 125 nm. (E, F) qRT-PCR analysis of mRNA expression levels of mitochondrial morphological genes encoding proteins on outer membrane (E) and inner membrane (F) in BMMSCs. (G) Western blot analysis of protein expression levels of Immt (Mitofilin) in BMMSCs. BMMSCs from 4-month-old SAMP6 mice were treated with either resveratrol (10 μM) or the DMSO (0.001%) solvent control. (H, I) qRT-PCR analysis of mRNA expression (H) and western blot analysis of protein expression (I) levels of Immt (Mitofilin) in osteogenic differentiation of BMMSCs from 4-month-old SAMR1 and SAMP6 mice. n = 3 per group. Data represent mean ± SD. *P < 0.05; NS, not significant (P > 0.05). Data were analyzed using ANOVA followed by Newman-Keuls post-hoc tests.

Figure 5

Mitofilin is indispensable for mitochondrial homeostasis and osteogenesis of BMMSCs. (A-D) Representative transmission electron microscopy images (A) and quantitative analysis of the ratio of mitochondrial inner membrane versus outer membrane (B) as well as mitochondrial average area (C) and number (D) per cell in BMMSCs. Bars: 125 nm (high magnification). (E, F) Rhodamine 123 detection of mitochondrial membrane potential of BMMSCs (E) with quantitative analysis of fluorescence intensity (F). Bars: 10 μm. (G, H) DCFDA detection of total ROS level in BMMSCs (G) with flow cytometric quantitative analysis of fluorescence intensity (H). Bars: 10 μm. (I) Quantitative analysis of ATP production versus ADP ratio in BMMSCs. (J) qRT-PCR analysis of mRNA expression levels of all 13 mtDNA-encoded mitochondrial complex subunits in BMMSCs. (K-M) Representative images of ALP and alizarin red staining (K) with quantification of ALP activity (L) and mineralization (M) in osteogenic differentiation of BMMSCs. Bars: 5 mm. (N) qRT-PCR analysis of mRNA expression levels of osteogenic marker genes in osteogenic differentiation of BMMSCs. BMMSCs from 4-month-old SAMR1 mice were transfected with either the shRNA for Immt (Mitofilin) or the negative control (a scrambled sequence, NC) by a lentiviral vector. n = 3 per group. Data represent mean ± SD. *P < 0.05; NS, not significant (P > 0.05). Data were analyzed using the two-tailed Student's t test.

Figure 6

Mitofilin mediates resveratrol-induced mitochondrial and osteogenic improvements of SAMP6 BMMSCs. (A-D) qRT-PCR analysis of mRNA expression levels of all 13 mtDNA-encoded mitochondrial complex subunits in complex I (A), complex III (B), complex IV (C) and ATP synthase (D) in BMMSCs. (E-G) Representative images of ALP and alizarin red staining (E) with quantification of ALP activity (F) and mineralization (G) in osteogenic differentiation of BMMSCs. Bars: 5 mm. (H, I) qRT-PCR analysis of mRNA expression (H) and western blot analysis of protein expression (I) levels of osteogenic marker genes in osteogenic differentiation of BMMSCs. BMMSCs from SAMP6 mice treated with resveratrol (10 μM) and the DMSO (0.001%) solvent control were transfected with either the shRNA for Immt (Mitofilin) or the negative control (a scrambled sequence, NC) by a lentiviral vector. n = 3 per group. Data represent mean ± SD. *P < 0.05; NS, not significant (P > 0.05). Data were analyzed using ANOVA followed by Newman-Keuls post-hoc tests.