Abstract

   Senescence of bone marrow mesenchymal stem cells (BMMSCs) induced by
   chronic oxidative stress is an important factor contributes to the
   postmenopausal osteoporosis (PMOP). Mitochondrial quality control takes
   a pivotal role in regulating oxidative stress and cell senescence.
   Genistein is a major isoflavone in soy products, which is best known
   for its ability to inhibit bone loss in both postmenopausal women and
   ovariectomized (OVX) rodents. Here we show that OVX-BMMSCs displayed
   premature senescence, elevated reactive oxygen species (ROS) level and
   mitochondria dysfunction, while genistein rescued these phenotypes.
   Using network pharmacology and molecular docking, we identified
   estrogen-related receptor α (ERRα) as the potential target of
   genistein. Knockdown of ERRα greatly abolished the anti-senescence
   effect of genistein on OVX-BMMSCs. Further, the mitochondrial
   biogenesis and mitophagy induced by genistein were inhibited by ERRα
   knockdown in OVX-BMMSCs. In vivo, genistein inhibited trabecular bone
   loss and p16^INK4a expression, upregulated sirtuin 3 (SIRT3) and
   peroxisome proliferator-activated receptor gamma coactivator one alpha
   (PGC1α) expression in the trabecular bone area of proximal tibia in OVX
   rats. Together, this study revealed that genistein ameliorates
   senescence of OVX-BMMSCs through ERRα-mediated mitochondrial biogenesis
   and mitophagy, which provided a molecular basis for advancement and
   development of therapeutic strategies against PMOP.

   Keywords: Mesenchymal stem cells, Senescence, Mitophagy,
   Estrogen-related receptor α, Osteoporosis

1. Introduction

   Postmenopausal osteoporosis (PMOP) is a systemic skeletal disorder
   characterized by low bone mass and increased fracture risk, which is
   triggered by estrogen deficiency during menopause [[43]1]. Premature
   senescence of stem cells is recognized as a critical pathogenesis of
   bone deteriorations in PMOP [[44]2]. Mechanistically, mitochondrial
   dysfunctions, in particular bioenergetic decline and reactive oxygen
   species (ROS) accumulation, are crucial mechanisms underlying stem cell
   senescence. Accordingly, approaches aimed at restoring mitochondrial
   homeostasis may provide promising opportunities to counteract PMOP
   [[45]3].

   Mitochondrial homeostasis is a sophisticated regulatory network that
   coordinates various cellular pathways, including the interplay between
   mitochondrial biogenesis and mitophagy. The tight coordination of these
   two opposing processes preserves mitochondrial function and cell
   homeostasis. Mitochondrial biogenesis provides the cell with newly
   synthesized mitochondria, thus preserves mitochondrial function and
   cellular homeostasis. Inhibition of mitochondrial biogenesis can lead
   to defective osteogenesis of bone marrow mesenchymal stem cells
   (BMMSCs) [[46]4]. Besides mitochondrial biogenesis, degradation of
   defective or superfluous mitochondria through mitophagy process also
   serves as another machinery for the maintenance of mitochondrial
   integrity and quality control [[47]5]. Defective mitophagy can lead to
   accumulation of damaged mitochondria [[48]6], which generate
   inefficient oxidative phosphorylation and further increases ROS
   production. Induction of mitophagy alleviates the damage caused by
   oxidative stress [[49]7]. Therefore, restoring the mitochondrial
   homeostasis of BMMSCs is possibly an effective method to against PMOP.

   Genistein, the principal soy phytoestrogen, has been proved to prevent
   bone loss in postmenopausal women [[50]8] and ovariectomized (OVX)
   rodents [[51]9,[52]10]. It was reported that genistein could mitigate
   mitochondrial dysfunctions in many pathological conditions, including
   neuroinflammation and aggregation of Aβ in Alzheimer's disease
   [[53]11]. Therefore, it is likely that genistein exert anabolic effect
   partially through modulating mitochondrial homeostasis to reduce
   oxidative stress, thus reverse premature senescence of OVX-BMMSCs
   during estrogen deficiency.

   In the present study, we systematically characterized the role of
   genistein in regulating the senescence of BMMSCs during estrogen
   deficiency. We determined that OVX-BMMSCs presented premature
   senescence phenotype compared to the Sham-BMMSCs, and genistein
   treatment greatly ameliorated cellular senescence in OVX-BMMSCs. Using
   integrated bioinformatic analysis, we identified estrogen-related
   receptor α (ERRα) as the potential target of genistein in PMOP.
   Remarkably, genistein-induced mitochondria biogenesis and mitophagy
   were largely prevented by transfection of ERRα siRNA. As a result, the
   anti-senescence effect of genistein in OVX-BMMSCs was abolished by ERRα
   knockdown. Collectively, these data demonstrate genistein exert
   anti-senescence effect on OVX-BMMSCs by restoring mitochondrial
   homeostasis in an ERRα-dependent manner, which provide further evidence
   for its use as a natural therapeutic agent for PMOP. It also suggested
   that the agonists of ERRα may be a viable therapeutic strategy to
   preserve bone mass in PMOP.

2. Materials and methods

2.1. Cell culture and reagents

   Twelve-week-old female Sprague-Dawley rats were subjected to either
   bilateral ovariectomy or sham surgery. Briefly, rats were anaesthetized
   and the bilateral ovaries were gently removed via a dorsal approach.
   For the Sham group, animals received the operation without removing the
   ovaries. Sixteen weeks after surgery, Sham-BMMSCs and OVX-BMMSCs were
   harvested from the bone marrow of the tibias and femurs from Sham rats
   and OVX rats, respectively. Sham-BMMSCs were maintained in DMEM medium
   plus 10% FBS (Gibco, Grand Island, NY, USA) and 1%
   penicillin-streptomycin (Gibco). OVX-BMMSCs were cultured in the same
   condition except that the DMEM medium is phenol red free (Gibco) to
   mimic the environment in vivo. Genistein and N-acetylcysteine (NAC)
   were obtained from Sigma (Sigma-Aldrich Inc., St. Louis, MO, USA) and
   bafilomycin A1 (Baf-A1) was purchased from CST (Cell Signaling
   Technology, Beverly, MA, USA). NAC is a classic antioxidant that is
   well known for its ability to minimize intracellular oxidative stress.
   Baf-A1, a vacuolar-type H^+-translocating ATPase (V-ATPase) inhibitor,
   was used as an autophagy inhibitor as it blocks the fusion of
   autophagosomes with lysosomes [[54]12].

   Antibodies were from various sources, including Abcam (anti-p21^CIP1,
   anti-TOM20, anti-PGC1α, anti-LC3B, anti-P62, HRP-conjugated goat
   anti-rabbit and FITC-conjugated goat anti-rabbit), CST (anti-p53,
   anti-CoxIV, anti-γH2AX, anti-β-actin, HRP-conjugated anti-rabbit and
   HRP-conjugated anti-mouse), Invitrogen (anti-p16^INK4a, anti-SIRT3 and
   anti-LepR), and Proteintech (anti-PGC1α and anti-Parkin).

2.2. Phenotypic characterization

   For phenotypic characterization, cells were stained with
   fluorochrome-conjugated monoclonal antibodies including CD45-FITC
   (Elabscience, E-AB-F1227C), CD31-FITC (Abcam, ab33858), CD90-FITC
   (Elabscience, E-AB-F1226C) and CD44-FITC (Elabscience, E-AB-F1225C).
   Corresponding mouse IgG1-FITC (Elabscience, E-AB-F09792C) and mouse
   IgG2a-FITC (Elabscience, E-AB-F09802C) were used as isotype control.
   The samples were incubated for 30 min in the dark and then washed for 2
   times. All samples were resuspended in 500 μl PBS and analyzed by flow
   cytometer (Beckman, CytoFLEX LX) and FlowJo software (Version 10.8.1,
   Tree Star, San Carlos, CA).

2.3. Small interfering RNA (siRNA) transfection

   ERRα siRNAs and negative control (NC) siRNAs were purchased from HanBio
   Technology Company (Shanghai, China) and the transfection was carried
   out according to the manufacturer's instruction. Briefly, cells were
   seeded in 6-well plate using growth medium without antibiotic one day
   before transfection. ERRα siRNA or NC siRNA were premixed with
   Lipofectamine RNAiMAX (Invitrogen) and then added into the cell medium
   with the final siRNA concentration of 100 nM. After 48-h incubation,
   the transfection medium was changed with fresh medium with or without
   genistein. The protein expression of ERRα in cells transfected with
   siRNA was examined by western blot analysis. The sequences of rat siRNA
   are as follows:

   ERRα: 5ʹ- GGCCUCCAAUGAGUGUGAGAU-3ʹ;

   Scrambled siRNA: 5ʹ-UUCUCCGAACGUGUCACGU-3ʹ.

2.4. Cell viability assay

   Cell viability was evaluated by cell counting kit-8 (Dojindo, Japan).
   Briefly, 3 × 10^3 cells were plated in 96 well plates and were
   incubated with various concentrations of genistein for 24 h, 48 h and
   72 h. After treatment, cells were incubated in 10% CCK-8 reagent for
   another 2 h. The optical density (OD) value was measured at 450 nm with
   a microplate reader (ELx800; BioTek Instruments, USA).

2.5. Colony-forming assays

   Cells (300 cells/well) were seeded into 6-well plates and cultured in
   conditioned medium for 10 days. Cells were fixed in 4% paraformaldehyde
   (m/v) for 15min at room temperature (RT). After rinsing with PBS, cells
   were stained with crystal violet solution (Sigma). The images were
   captured by microscope and the colonies (aggregates≥50 cells) were
   counted.

2.6. Senescence β-galactosidase (β-Gal) staining

   To assess cell senescence, β-Gal activity was determined by using β-Gal
   Staining Kit (Beyotime, Institute of Biotechnology, China). Briefly,
   cells were fixed in fixation buffer for 15 min at RT, then washed and
   incubated with staining solution at 37 °C overnight. The number of blue
   cells positive for SA-β-Gal was expressed as % of total cell number
   (100 cells counted for each condition, three independent experiments).

2.7. Immunofluorescence staining

   Cells were seeded onto gelatin-coated glass slides and treated with
   conditioned medium for 48 h. After removing the medium, the cells were
   fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.2%
   Triton X-100 for 10min, and then blocked with goat serum for 1 h. For
   protein detection and localization, the cells were incubated with
   specific primary antibodies (anti-γH2AX, 1:400; anti-Parkin, 1:200;
   anti-TOM20, 1:200) overnight. All samples were then washed 3 times and
   incubated with fluorescent secondary antibody (1:1000, Abcam) for 2 h.
   DAPI was used for nucleus counter staining. The photographs were
   acquired by using a Nikon fluorescence microscope. The γH2AX intensity
   was divided by the DAPI intensity. Three random fields of view were
   included into the measurement for each slide, and the assays were
   repeated for three biological replicates.

2.8. Mitochondrial DNA copy number assay

   Mitochondrial DNA (mtDNA) copy number was determined by quantitative
   real-time PCR as described [[55]13]. Briefly, total DNA was extracted
   and purified from cells using a DNeasy Blood & Tissue kit (Qiagen,
   Hilden, Germany). Quantification of mtDNA was performed by use of the
   ratio of mtDNA marker mitochondrial NADH dehydrogenase 1 (mt-ND1)
   (Forward: 5′-CGTTGCCCAAACCATCTCT-3′, Reverse: 5′-CTATTGGTCAGGCGGGGA-3′)
   to nuclear DNA marker β-actin (Forward: 5′-
   TTGTCACCAACTGGGACGATATGG-3′, Reverse: 5′- GGGGTGTTGAAGGTCTCAAACATG-3′).
   The relative mtDNA copy number was measured by normalization of the
   crossing points in quantitative PCR curves between ND1 and β-actin
   genes using the RelQuant software (Roche Applied Sciences).

2.9. ROS measurements

   Cellular ROS level was measured by a DCFDA fluorescent probe
   (Beyotime). Briefly, cells were wash with HBSS twice and incubated with
   serum-free DMEM containing 10 μM DCFDA at 37 °C for 20 min. The
   photographs were captured by a Nikon fluorescence microscope. ROS
   generation was determined by using a FACSCalibur flow cytometer
   (Beckman, CytoFLEX LX). The data from flow cytometry were analyzed
   using FlowJo software, and mean fluorescence intensity (MFI) in each
   group was recorded and analyzed. When indicated, 5 mM NAC was used as
   an antioxidant positive control.

2.10. Mitochondrial superoxide Quantification

   Cells were incubated with 5 μM MitoSOX Red (Invitrogen) for 10 min,
   then washed with warm HBSS to remove the excess dye. To visualize the
   stained cells, random fields in each group were captured by using a
   Nikon fluorescence microscope. To quantify the MitoSOX Red fluorescence
   intensity, cells were collected and sorted by FACSCalibur flow
   cytometer (Beckman). The data from flow cytometry were analyzed using
   FlowJo software, and the mean fluorescence intensity (MFI) in each
   group was recorded and analyzed.

2.11. ATP content and mitochondrial membrane potential detection

   ATP content of cells was determined with the ATP assay kit (Abcam,
   ab83355) according to manufacturer's protocol. Briefly,1 × 10^6 cells
   were resuspended in 100 μL of ATP assay buffer, then homogenized and
   centrifuged for 5 min at 4 °C. The supernatant was collected and added
   to plate at 50 μL/well. Add 50 μL ATP reaction mix to each well,
   incubate the plate at room temperature for 30 min, then measure output
   on a microplate reader at OD 570 nm. Subtract the mean value of the
   blank from all standards and sample readings. The sample values were
   calculated from the standard curve and normalized to the control group
   in each individual experiment.

   For measuring mitochondrial membrane potential, a JC-1 kit (Beyotime)
   was used. JC-1 is cationic dye that exhibit potential-dependent
   accumulation in mitochondria, indicated by a fluorescence emission
   shift from green to red. Mitochondrial depolarization is indicated by a
   decrease in the red/green fluorescence intensity ratio. Cells were
   incubated with JC-1 dye for 20 min at 37 °C in the dark. Thereafter,
   cells were either assessed by fluorescence microscope or harvested and
   analyzed by flow cytometry. The data from flow cytometry were analyzed
   using FlowJo software, and median PE and FITC fluorescence were used to
   calculate a red/green ratio as an index of mitochondrial membrane
   potential [[56]14].

2.12. Oxygen consumption rate (OCR)

   The measurement of OCR in cells were performed using Seahorse XF96
   analyzer (Agilent Technologies, Santa Clara, CA) following
   manufacturer's protocol. Briefly, OVX-BMMSCs were plated at density of
   10,000 cells/well in a Seahorse cell culture microplate and transfected
   with siNC/siERRα. After 48 h transfection, the media is replaced with
   culturing medium with/without genistein for another 72 h. On the day of
   measurement, the medium was changed to pre-warmed Seahorse assay
   medium, and OCR determined using the Seahorse XF Cell Mito Stress Kit
   (Agilent). After recording 3 total cellular respiration measurements in
   unstimulated cells, oligomycin (1.5 μM) was added to inhibit
   mitochondrial ATP synthase and measure the decrease in the OCR that is
   linked to ATP turnover. To determine the maximal respiration potential
   of the cells, FCCP (1 μM; an uncoupler of oxidative phosphorilation)
   was used. The amount of nonmitochondrial oxygen consumption was
   determined by inhibiting the respiratory chain activity with antimycin
   A and rotenone cocktail (0.5 μM). After completion, cells were lysed
   with RIPA buffer (Beyotime), and OCR was normalized to protein content
   determined using a BCA protein assay kit (Beyotime) according to the
   manufacturer's instructions.

2.13. Transmission electron microscopy (TEM) analysis

   Cells were washed with PBS (PH 7.4) and primarily fixed in 2.5%
   glutaraldehyde for 24 h. The samples were post-fixed in 1% OsO[4] for
   1 h at RT, then progressively dehydrated through graded ethanol/acetone
   solutions and embedded in epoxy resin. Finally, ultrathin sections
   (70 nm) were prepared and stained with 2% uranyl acetate followed by
   lead citrate. The images of cell ultrastructure were captured with a
   transmission electron microscope (JEOL, Tokyo, Japan). The experiments
   were repeated for three biological replicates.

2.14. Live cell imaging microscopy

   Cells were loaded with green-fluorescing mitochondrial dye Mito-Tracker
   Green (MTG, 20 nM) (Invitrogen) for 30min at 37 °C, then washed 2 times
   with fresh complete growth medium and incubated with red-fluorescing
   LysoTracker Red (LTR, 50 nM) (Invitrogen) for 30min at 37 °C. After MTG
   and LTR had been loaded, confocal images were acquired by using a
   confocal laser scanning microscope (TCS SP8, Leica Microsystems,
   Germany). MTG was excitated at 490/516 nm and LTR was excitated at
   577/590 nm. Co-localization of both signals is counted as a mitophagy
   event. The Pearson's colocalization coefficient between MTG and LTR
   fluorescence intensities was determined in 9 cells from three
   independent experiments using Image J software [[57]13].

   For functional mitochondria staining, cells were loaded with
   Mito-Tracker Red CMXRos (MTR, 20 nM) (Invitrogen) for 30min at 37 °C,
   then washed 2 times with fresh complete growth medium. Confocal images
   were acquired by using a confocal laser scanning microscope (Leica).
   MTR was excitated at 579/599 nm. The stained cells were also sorted by
   FACSCalibur flow cytometer (Beckman). The data from flow cytometry were
   analyzed using FlowJo software, and the mean fluorescence intensity
   (MFI) in each group was recorded and analyzed.

2.15. Total RNA extraction and quantitative real-time PCR analysis

   Targeted gene expression analyses were performed by rt-qPCR as
   described [[58]15]. Briefly, total RNA was extracted with Trizol
   reagent (Invitrogen, Carlsbad, CA), followed by cDNA synthesis using
   Reverse Transcription Kit (TaKaRa, Japan) according to the
   manufacturer's instructions. qRT-PCR were performed on a Roche Light
   Cycler 480 (Roche, Mannheim, Germany) using the SYBR Green PCR Master
   Mix (TaKaRa, Japan). Fold changes of mRNA were calculated by the
   2^−ΔΔCt method after normalization to the expression of β-actin (ACTB).
   Sequences of the primers are listed in [59]Supplementary Table S1.

2.16. Western blotting

   The cells were dissociated with RIPA buffer (Beyotime) containing
   protease inhibitor and phosphatase inhibitors (Sigma). After
   concentration measurement, 20 μg of protein was loaded onto 4–20%
   SDS-PAGE gel and transferred to a PVDF membrane (Millipore, Billerica,
   MA, USA). The membrane was blocked by 5% non-fat milk for 2 h followed
   by incubated with primary antibody at 4 °C overnight. Subsequently, the
   blots were incubated for 1 h at RT with an HRP-conjugated secondary
   antibody (CST), and visualized by chemiluminescence detection system
   (Millipore). Quantitative analysis of the Western blot was carried out
   by using Image J software, and band intensity was normalized to
   β-actin.

   The primary antibodies used were, anti-p21^CIP1 (1:1000, Abcam,
   ab109199) anti-TOM20 (1:1000, Abcam, ab186735), anti-PGC1α (1 μg/ml,
   Abcam, ab106814), anti-LC3B (1:2000, Abcam, ab192890), anti-P62
   (1:1000, Abcam, ab56416), anti-p53 (1:1000, CST, #2524), anti-CoxIV
   (1:1000, CST, #4850), anti-β-actin (1:1000, CST, #8457), and anti-SIRT3
   (1:1000, Invitrogen, PA5-96406).

2.17. Drug-target interaction prediction

   Drug–target network (DTN) from genistein and targets associations in
   postmenopausal osteoporosis was constructed and analyzed by the
   DrugBank 4.0 database. Then, we mapped the primary target proteins into
   the online STRING database to generate protein-protein interaction
   (PPI) network generation. Interaction pairs with overall score above
   0.9 were recorded. To characterize the biochemical pathways and
   functions linked to genistein, GO (Gene Ontology) enrichment analysis
   and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment
   analysis were carried out.

2.18. Molecular docking analysis

   The chemical structure of genistein was obtained from the PubChem
   database, and genistein were imported into Schrodinger for 3D format
   conversion and energy minimization. The protein structure of ERRα was
   obtained by SwisS-Model online server and processed in Maestro11.9.
   Schrodinger's Protein Preparation Wizard was used to process the
   protein structure, remove the crystal water, replenish the missing
   hydrogen atoms, repair the missing bond information, repair the missing
   peptide, and finally minimize the energy of the Protein
   [[60]16,[61]17]. Then, Molecular docking studies using ERRα with
   genistein was performed by the Glide module in Schrodinger Maestro
   software. Finally, the screening and analysis of docking results were
   carried out by SP.

2.19. Animals and drug treatment

   All animal protocol was critically reviewed and approved by the
   institutional review board of Shanghai Jiao Tong University School of
   Medicine (institutional review board no. SH9H-2020-A704-1). This study
   conformed to the ARRIVE (Animal Research: Reporting In Vivo
   Experiments) guidelines for animal studies.

   A total of 60 female Sprague-Dawley rats, mean weight 220 ± 25 g, were
   obtained at 3-month of age from the Ninth People's Hospital Animal
   Center (Shanghai, China). Rats were maintained under the following
   environmental conditions: temperature: 25 ± 3 °C; humidity: 50 ± 10%;
   illumination: light/dark cycle 12/12; free access to water and a diet
   low in phytoestrogen content. Following 2 weeks of adaptation, 30 rats
   were received operation to establish the OVX-OP model and 30 rats were
   received sham operation.

   Eight weeks after surgery, rats received the following treatments with
   15 rats in each group: (1) Sham + VEH group: oral saline solution
   treatment once daily; (2) Sham + GEN group: oral genistein (50 mg/kg
   body weight, Sigma-Aldrich, MO, USA) treatment once daily. (3)
   OVX + VEH group: oral saline solution treatment once daily; (4)
   OVX + GEN group: oral genistein (50 mg/kg body weight) treatment once
   daily.

   After being fed for 8 weeks, blood was withdrawn before the rats were
   sacrificed, and serum was then prepared. Rats were sacrificed by
   cervical dislocation under halothane anesthesia. Femurs and tibias were
   collected for further analysis.

2.20. Measurement of serum MDA

   The activity of MDA in serum was measured by using a lipid peroxidation
   MDA assay kit (Beyotime) according to the manufacturer's instructions.
   Briefly, the serum was incubated with reagent in each reaction tube at
   100 °C for 15 min. After centrifuged at 4000 rpm for 10 min, the
   supernatants were collected and measured for absorbance at 532 nm with
   a microplate reader.

2.21. Bone biomechanical test

   The femurs were stored frozen at −20 °C before biomechanical test.
   After thawing, the right femur mid-shaft was taken for the three-point
   bending assay by using electronic universal testing machine (Shenzhen
   Reger Instrument Co. Ltd., China) according to Ma et al.'s [[62]18]
   description. A load was vertically applied to the femoral mid-shaft,
   with a displacement speed of 0.1 mm/s and span distance at 20 mm until
   the femoral shaft is fractured. The left femoral neck was tested by
   mounting the proximal half of the femur vertically in a chuck and
   applying a downward force on the femoral head until failure. The
   ultimate load of the femurs was recorded and analyzed.

2.22. Micro-computed tomography (Micro-CT)

   Micro-CT scanning was performed by using a micro-CT system (μCT-80,
   Scanco Medical, Bassersdorf, Switzerland) on proximal tibias before
   decalcification. To visualize the inside of the proximal tibia,
   two-dimensional (2-D) images at their most central part were obtained.
   Trabecular bone was selected as region of interests (ROI) and the
   following parameters of ROI were analyzed: bone mineral density (BMD),
   bone volume/total volume (BV/TV), trabecular thickness (Tb.Th) and
   trabecular separation (Tb.Sp).

2.23. Histological analysis

   Decalcification of specimens was carried out with 10% EDTA with
   constant shaking for 8 weeks. After decalcification, the specimens were
   embedded in paraffin and cut into 5-μm-thick sections. Three sections
   of the most central part of the proximal tibia were selected and
   underwent H-E staining. For immunohistochemistry, sections were
   deparaffinized, rehydrated, and incubated with primary antibody
   (anti-p16^INK4a, 5 μg/mL, Invitrogen) at 4 °C overnight after antigen
   retrieval. The sections were then washed in PBS and incubated with
   secondary antibody (HRP-conjugated goat anti-rabbit, 1:1000, Abcam),
   and stained with DAB kit. The sections were observed under a light
   microscope.

   For immunofluorescence staining, sections were incubated with a primary
   antibody (anti-SIRT3, 1:100, Invitrogen) at 4 °C overnight and then
   with a secondary antibody (FITC-conjugated goat anti-rabbit, 1:1000,
   Abcam) for 1 h at 37 °C. Afterwards, slides were stained with DAPI and
   mounted with an anti-fade reagent (Invitrogen). Confocal images of
   slides were obtained by using a confocal microscope (Leica).

   For peroxisome proliferator-activated receptor gamma coactivator one
   alpha (PGC1α) and leptin receptor (LepR) immunofluorescence staining,
   sections were blocked with 10% goat serum (Gibco) and then incubated
   with primary antibodies (anti- PGC1α, 1:50, Proteintech; anti-LepR,
   1:50, Invitrogen) at 4 °C overnight and with secondary antibodies for
   1 h at 37 °C. Slides were then stained with DAPI and mounted with an
   anti-fade reagent (Invitrogen). The images were captured by a Zeiss
   LSM710 confocal microscope (Carl Zeiss).

   Image J software was used for quantification of IHC and IF. For
   quantification of p16^INK4a staining, three random fields of view per
   specimen were taken, and the average values were taken as the data
   point for each specimen. The SIRT3 intensity was divided by the DAPI
   intensity, and the PGC1α intensity was divided by the LepR intensity.
   Three random fields of view were included into the measurement for each
   specimen, and nine specimens in each group were selected for
   immunohistochemistry and immunofluorescence staining. All data was
   analyzed blinded.

2.24. Statistical analysis

   Data were presented as means ± SD. At least three samples or
   independent experiments were performed with all the assays. All
   statistical analyses were performed using the GraphPad prism (Version
   9.0, GraphPad Software, San Diego, CA). Normal distribution of the data
   was verified using Shapiro-Wilk test and the equality of variances was
   verified by Levene's test. P values were calculated by unpaired
   Student's t-test, or one-way ANOVA for samples following normal
   distribution. For ANOVA, Tukey's post-test was used to compare
   individual groups. Mann-Whitney, or Kruskal-Wallis tests were used when
   samples didn't follow a normal distribution. For Kruskal-Wallis test,
   Dunn's multiple comparisons test was used for post-hoc analysis. P
   value < 0.05 was taken as statistical significance.

3. Results

3.1. Genistein alleviated the senescence of OVX-BMMSCs

   Primary cultured BMMSCs were positive for surface markers (CD44 and
   CD90) and negative for hematopoietic cell markers (CD31 and CD45)
   ([63]Fig. 1A). Cell viability assay showed that genistein had no cell
   toxicity when used at concentrations ranging from 10^−3 to 1 μM
   ([64]Fig. 1B). The expression of phosphorylated H2A histone family
   member X (γH2AX), a marker of DNA damage, was upregulated in the
   OVX-BMMSCs versus Sham-BMMSCs ([65]Fig. 1C and D). Consistently,
   OVX-BMMSCs displayed senescence phenotype, as shown by the reduced
   colony forming ability ([66]Fig. 1E and F) and increased SA-β-gal
   positive cells ([67]Fig. 1E, G). Then, we explored the effect of
   genistein on senescence of OVX-BMMSCs at concentration of 1 μM and
   10^−2μM, as these concentrations have been identified as the optimal
   concentrations in previous study [[68]19]. Administration of genistein
   could increase the number and size of colonies, and downregulate the
   SA-β-gal and γH2AX positive cells ([69]Fig. 1C–G). We also analyzed the
   mRNA and protein expressions of senescence related marker [[70]20] p53,
   p16^INK4a and p21^CIP1, and found that the OVX-induced upregulation of
   p53, p16^INK4a and p21^CIP1 in BMMSCs were inhibited by genistein
   treatment ([71]Fig. 1H and I). Collectively, these data indicated that
   the estrogen-deficiency induced premature senescence could be
   alleviated by genistein treatment in BMMSCs.

Fig. 1.

   [72]Fig. 1
   [73]Open in a new tab

   Genistein alleviated cellular senescence of OVX-BMMSCs. (A) Flow
   cytometric analysis for cell surface markers of rat BMMSCs (n = 3). (B)
   Cell viability assay of cells treated by escalating doses of genistein
   (0–10 μM) for 24 h, 48 h, and 72 h (n = 3). (C, D) Representative
   images of γH2AX staining and quantitative assessment of the γH2AX
   positive cells (n = 3). DAPI-labeled nuclei are in blue and γH2AX is
   stained in green. Cells were treated by genistein for 3 days. Scale
   bar, 50 μm. (E, F and G) The representative images and quantitative
   analysis of colony forming assay and SA-β-gal staining (n = 3). Scale
   bar, 500 μm. (H, I) The mRNA and protein expression of senescence
   related marker p53, p16^INK4a and p21^CIP1 of Sham-BMMSCs and
   OVX-BMMSCs. Cells were treated by genistein for 3 days, when indicated.
   Statistical analysis of expression of each marker was adjusted to
   β-actin (n = 3). Each value is mean ± SD. Significance was calculated
   using a one-way ANOVA followed by the Tukey's post-hoc test (B, D, G, H
   and I). Kruskal-Wallis test followed by Dunn's multiple comparisons
   test was used for F. *p < 0.05 and **p < 0.01 compared to the Sham
   group. #p < 0.05 and ##p < 0.01 compared to the OVX group. (For
   interpretation of the references to color in this figure legend, the