Dendrobium officinale polysaccharides regulate age‐related lineage commitment between osteogenic and adipogenic differentiation

Abstract Objectives Excessive oxidative stress and diminished antioxidant defences could contribute to age‐related tissue damage and various diseases including age‐related osteoporosis. Dendrobium officinale polysaccharides (DOPs), a major ingredient from a traditional Chinese medicine, have a great potential of antioxidative activity. In this study, we explore the role of DOP in age‐related osteoporosis that remains elusive. Materials and methods Oxidative stimulation and DOP were used to treat bone marrow mesenchymal stem cells (BMSCs), whose lineage commitment was measured by adipogenic‐ and osteoblastic‐induced differentiation analysis. The oxidative stress and antioxidant capacity of BMSCs under the treatment of DOP were analysed by the level of MDA, SOD. Related mechanism studies were confirmed by qRT‐PCR, Western blotting and siRNA transfection. DOP was orally administrated in aged mice whose phenotype was confirmed by micro‐CT, immunofluorescence, immunochemistry and calcein double‐labelling analysis. Results Dendrobium officinale polysaccharide treatment markedly increased osteogenic differentiation of BMSCs, while inhibiting adipogenic differentiation. In vitro, DOP could rescue H2O2‐induced switch of BMSCs differentiation fate. However, this effect was abolished in BMSCs when interfered with Nrf2 siRNA. Furthermore, administration of DOP to aged mice significantly increased the bone mass and reduced the marrow adipose tissue (MAT) accompanied with decreased oxidative stress of BMSCs. Conclusions Our study reveals that DOP can attenuate bone loss and MAT accumulation through NRF2 antioxidant signalling, which may represent as potential therapeutic agent for age‐related osteoporosis.

age-related diseases, such as osteoporosis, osteoarthritis, diabetes, cardiovascular diseases and neurodegeneration. [3][4][5][6][7][8] It has been reported that oxidative stress is recognized as an independent factor involved in age-related bone loss and could be associated with insufficient number of osteoblast and bone formation rate. 3,9, 10 Domazetovic et al demonstrated that oxidative stress impaired bone formation and subsequently contributed age-related osteoporosis via attenuating osteoblastic differentiation. 11 During ageing, bone marrow stem cells (BMSCs) tend to differentiate into adipocytes rather than osteoblasts, which subsequently lead to progressive MAT accumulation and bone loss. 10,12,13 The imbalance between bone and fat in age-related osteoporosis has been reported to be associated with an increasingly proinflammatory tissue environment with mounting oxidative stress. [14][15][16] Conversely, peroxisome proliferator-activated receptor coactivator 1-a (PGC-1a), with property of antioxidative defence, had an anti-osteoporotic role though promoting osteogenesis and inhibiting adipogenesis. 14 Dendrobium officinale, a traditional Chinese health medicine, has received much attention in recent years due to its pharmacological properties. 17,18 Dendrobium officinale polysaccharides (DOPs) are a major ingredient in Dendrobium officinale, which was reported to possess a wide range of potential effects including antioxidative, anti-inflammatory, immunomodulation, anticancer, anti-obesity, antihypertensive, hypoglycaemic and neuron protection activity. [19][20][21][22] Previous studies have shown that Dendrobium officinale extract can prevent the osteoporosis in ovariectomized mice. 23 Moreover, ZHAO et al revealed that DOP has a great role in regulating cell fate of BMSCs. 24 It could inhibit adipogenic differentiation in Rat BMSCs by deregulating adipogenesis-related gene expression of PPARγ, LPL and FABP4. 24 However, the role of DOP in age-related bone loss and lineage commitment of BMSCs is still unclear.
Here, the present study demonstrates that DOP, as an antioxidative reagent, can attenuate oxidative stress damage and subsequently regulate age-related BMSCs lineage commitment shift whose effects are related to Nrf2-mediated antioxidative response.
Taken together, our study provides potential therapeutic effect of DOP in age-related osteoporosis.

| Mice
The 15-month-old mice were orally administrated with normal saline (NS) and DOP (150 mg/kg), respectively, once daily for 3 months.

| Mouse BMSCs isolate and cell culture
Mouse BMSCs isolation was described as before. 13 Briefly, the cells were incubated with Sca-1, CD29, CD45 and CD11b (BioLegend) after bone marrow cells were flushed from femora bone marrow cavity. Then, we sorted out Sca-1 + CD29+CD45−CD11b-BM-MSCs through flow cytometry. Next, cultured the cell with low-glucose DMEM, 100 U/mL of penicillin/streptomycin and 10% FBS in a humidified atmosphere of 95% air and 5% CO 2 at 37 °C.

| Human bone marrow collection
We obtained the written informed consent from all participants before collecting bone marrow. The age of the participants was ranged from 20 to 79 years underwent hip replacement. All clinical bone marrows were approved by the Ethics Committee of the Xiangya Hospital of Central South University, and this study conformed to recognized standards.

| Detection of cell viability
Cell viability was assessed by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay according to manufacturer's instructions.
The absorbance was measured at 570 nm using a Multiskan™ GO microplate reader (Thermo Fisher Scientific).

| Osteogenic differentiation and mineralization assay
BMSCs were cultured in 6-well plates at a density of 2.5 × 10 6 cells per well with osteogenesis induction medium (50 μg/mL ascorbic acid, 1 µmol/L dexamethasone and 5 mM β-glycerophosphate) for 21 days. 25 For ALP staining, we fixed the cell for 5 minutes with 10% formaldehyde, then incubated them for 2 hours at 37°C with ALP incubation buffer (0.2 g barbital sodium, 0.4 g magnesium sulphate, 0.2 g calcium chloride and 0.3 g beta-glycerophosphate). Next, washed cells with 2% calcium chloride and incubated with 2% cobaltous nitrate for 5 minutes, then incubated them with 1:80 ammonium sulphate for 10 seconds.
Finally, the cells were observed by microscope (Leica) and statistically calculated by Photoshop. We performed and quantified Alizarin red staining (Sigma-Aldrich) to evaluate the cell matrix mineralization and observed by microscope (Leica) according to previous protocol. 13

| Adipogenic differentiation
BMSCs were cultured with adipogenesis induction medium (DMEM containing 10% FBS, 0.5 mmol/L 3-isobutyl-1-methylxanthine, 5 μg/ mL insulin and 1 μmol/L dexamethasone) for 14 days. We performed oil red O staining to detect mature adipocytes and quantified oil red released from the cell into the isopropanol solution by spectrophotometry at 540 nm according to previous protocol. 13

| Western blot and qRT-PCR analysis
We performed Western blotting as previously described. 26 The primary antibodies, NRF2 (ab62352; Abcam) or α-tubulin (11224-1-AP; Proteintech), were incubated overnight, then incubated with appropriate HRP-conjugated secondary antibodies for 1 hour at room temperature. The blots were visualized using ECL detection reagents. For qRT-PCR analysis, total RNA from cultured cells was extracted using Trizol reagent (Invitrogen) and then performed qPCR using PrimeScript RT reagent Kit (Takara) and SYBR Green PCR Master Mix (Takara). Primer sequences are listed in Tables S1 and S2.

| H 2 O 2 treatment and antioxidant activity
Human MSCs were pre-exposed to 100 μmol/L H 2 O 2 for 2 hours.
The level of SOD and MDA in BMSCs was determined by MDA assay kit and SOD assay kit (Nangjing Jiancheng Bioengineering Institution) following the instruction of manufacturer.

| Micro-CT analysis
The femurs were dissected from mice and fixed with 4% paraformaldehyde overnight, then scanned and analysed with high-resolution micro-CT (Skyscan 1172, Bruker MicroCT) as previously described. 13 We selected the region of interest for analysis as 5% of femoral length below the growth plate. Trabecular bone volume per tissue volume (Tb. BV/TV), trabecular number (Tb. N), trabecular separation (Tb. Sp) and trabecular thickness (Tb. Th) were measured.

| Immunohistochemistry analysis
Freshly, femora were dissected from mice, fixed overnight at 4℃ with 4% paraformaldehyde, decalcified in 10% EDTA (pH 7.4) for 21 days and then embedded in paraffin. Bone sections (4 μm thick, longitudinally oriented) were incubated with primary antibody against osteocalcin (Takara, M173) overnight at 4°C. Subsequently, an HRP-streptavidin detection system (Dako) was used to detect the immunoactivity, followed by counterstaining with haematoxylin (Sigma-Aldrich). Sections incubated with polyclonal rabbit IgG (R&D Systems) served as negative controls. Four randomly selected visual fields in the distal metaphysis of the femur were measured to test the number per millimetre of adjacent bone surface (Nmm -1 ) in trabecular bone.

| Calcein double labelling
We injected mice intraperitoneally with 0.08% calcein (Sigma-Aldrich, 20 mg/kg bw) 8 and 2 days before euthanasia. Calcein double labelling in undecalcified bone slices was observed under a fluorescence microscope. Four randomly selected visual fields in the distal metaphysis of the femur were measured to test trabecular bone formation in femora.

| Statistics
Data are presented as mean ± SD. We used two-tailed Student's t test for 2-group comparison and one-way ANOVA for comparison within multiple groups. All experiments were conducted at least three times, and representative experiments were shown. P < 0.05 was considered as significant difference. two key markers of adipocyte differentiation, were both significantly inhibited by DOP in a dose-dependent manner ( Figure 1H).

| DOP stimulated osteoblast differentiation and inhibited adipocyte differentiation in BMSCs
Together, these results suggested that DOP stimulated osteoblastic differentiation and suppressed adipocytic differentiation of BMSCs.

| DOP regulated differentiation of BMSCs through activating Nrf2 signalling
DOP has been reported to be associated with antioxidative activity through NRF2 signalling pathway. 27

| DOP restored oxidative stress-induced damage during osteoblastic and adipogenetic differentiation
To further confirm the antioxidative activity of DOP, we treated

| NRF2 expression level in human BMSCs was decreased during ageing
Ageing is always accompanied with increased oxidative stress. We isolated and cultured human BMSCs (hBMSCs) from male subjects at different ages. Compared to young group, the old group represented a remarkable decrease in SOD activity which indicated antioxidative capability ( Figure 4A) and an increase in MDA level which indicated oxidative stress level ( Figure 4B). These data implied an elevated level of oxidative stress in hBMSCs during ageing. Specifically, the expression of NRF2 was notably downregulated with ageing both at mRNA level and protein level ( Figure 4C,D). Alternatively, qRT-PCR analysis revealed that antioxidative enzymes were correlated with NRF2 ( Figure 4C). These results support that antioxidant NRF2 signalling plays an important role in the ageing process.

| DOP treatment attenuated bone loss and MAT accumulation in aged mice
To investigate the therapeutic effects of DOP in oxidative stress and age-related osteoporosis in vivo, we treated the 15-month-old mice with normal saline (NS) and DOP, respectively. DOP was orally administrated with the dose of 150 mg/kg once daily for 3 months. Notably, DOP significantly decreased the oxidative stress damage (Figure 5A,B) and increased Nrf2 expression in vivo ( Figure 5C,D). qRT-PCR analy-

| D ISCUSS I ON
Age-related bone loss is becoming a growing public health problem as the progressive ageing population. 1-3 It urgently requires us to find some potential therapeutic targets and agents to prevent the development of bone loss. [29][30][31] In this study, we confirmed the potential therapeutic target of NRF2 in age-related bone loss and the protective role of DOP in attenuating the oxidative stress-impaired osteoblast differentiation. It is well established that oxidative stress is prerequisites for the progression in skeletal ageing and osteoporosis. 3,5,32 Extensive researches suggested that oxidative stress accumulation tified that the deficiency of NRF2 antioxidant pathway was a driver mechanism in premature ageing disorder Hutchinson-Gilford progeria syndrome (HGPS), and the impairment of NRF2 transcriptional activity would cause oxidative stress and related progeroid phenotypes including bone loss. 33 These results indicated that NRF2 antioxidant activity was dispensable for age-related diseases.
NRF2 is regarded as the master regulator of the cellular antioxidant response and a therapeutic target of oxidative-mediated diseases. [34][35][36] It is also a controversial player in bone metabolism. 28,[37][38][39] Preliminary reports suggested overexpression of Nrf2 enhanced the potential of BMSCs to differentiate into osteogenic lineage and inhibited the potential to differentiate into adipogenic lineage. 34,38 Moreover, the deletion of Nrf2 in mice leads to a lower bone mass and an bone microarchitecture. 40,41 In our study, we utilized hBMSCs from different age subjects to detect the relationships among age-related BMSCs. It indicated Nrf2 was a key factor in BMSCs lineage commitment. However, some studies suggested that Nrf2 was a detrimental factor in bone haemostasis. 37,40,42 We speculated that the previous reported inconsistent effects were associated with some confounding factors, such as different sex, age, genetic background and even cross communications between cells in skeletal system or communication with other organs in the global Nrf2 knockout mice. 37,43 DOP is a major ingredient in Dendrobium officinale and known with its antioxidant activity. 44 Some work suggested the protective role of Dendrobium officinale in bone mass and natural ageing-induced premature ovarian failure. 45 In consistent with previous protective results, our study found that DOP remarkably increased osteogenesis and decreased adipogenesis in BMSCs, which could contribute to prevent age-related bone loss. Moreover, some studies showed that DOP had a positive effect on activating Nrf2 signalling. 27 In this paper, we demonstrated that BMSCs showed deficient osteogenesis and enhanced adipogenesis when exposure to oxidative stress, and the treatment of DOP could reverse this effect. However, this effect rescued by DOP treatment was absent in Nrf2-knockdown BMSCs. Moreover, we also validated its anti-osteoporosis activity assessed by remarkably restored bone loss and MAT accumulation in the treated mice, which were accompanied with upregulated defence against oxidative stress.
Together, these findings imply that DOP controls age-related lineage commitment of BMSCs and bone-fat balance by activating antioxidant NRF2 signalling ( Figure 6). Our study demonstrates a rationale therapeutic target and a potential anabolic agent in age-related bone loss.

CO N FLI C T O F I NTE R E S T S
The authors have declared that no conflict of interest exists.

Hui Peng
https://orcid.org/0000-0002-7673-3919 F I G U R E 6 Schematic illustration of DOP-regulating age-related osteogenesis and adipogenesis in bone marrow via activating NRF2 signalling. DOP has a positive effect on restoring the ageinginduced oxidative stress and imbalanced fate shift between adipocytes and osteoblasts in BMSCs through the antioxidant NRF2 signalling