Effects of Dendrobium officinale polysaccharide on adipogenic differentiation of rat bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (BMSCs) are the pluripotent adult stem cells, which can differentiate into osteoblasts, adipocytes, nerve cells, cartilage cells and muscle cells under certain induction conditions (Kopen et al., 1999; Tagami et al., 2003; Baksh et al., 2007; Izadpanah et al., 2008). In recent years, BMSCs are closely related to the occurrence and development of osteoporosis. The decrease of bone mass is always accompanied with the increase of adipose cells in bone marrow cavity (Carbonare et al., 2009). Therefore, seeking an effective way to inhibit the differentiation of BMSCs into adipogenic cells has become a research target for prevention and treatment of osteoporosis.


Introduction
Bone marrow mesenchymal stem cells (BMSCs) are the pluripotent adult stem cells, which can differentiate into osteoblasts, adipocytes, nerve cells, cartilage cells and muscle cells under certain induction conditions (Kopen et al., 1999;Tagami et al., 2003;Baksh et al., 2007;Izadpanah et al., 2008).In recent years, BMSCs are closely related to the occurrence and development of osteoporosis.The decrease of bone mass is always accompanied with the increase of adipose cells in bone marrow cavity (Carbonare et al., 2009).Therefore, seeking an effective way to inhibit the differentiation of BMSCs into adipogenic cells has become a research target for prevention and treatment of osteoporosis.
Dendrobium officinale is a precious medicinal plant recorded in the Chinese pharmacopeia.It is an epiphytic and perennial herb, and is mainly distributed in the south areas of China.Dendrobium officinale has many kinds of main active ingredient in which polysaccharide is the main ingredient (Chen et al., 2012).Previous studies demonstrate that Dendrobium officinale polysaccharide (DDP) possesses antioxidant, immunomodulatory and anti-hypoglycaemic activities (Pan et al., 2014;Cai et al., 2015).It is found that, some polysaccharides from herb can prevent the osteoporosis (Yang et al., 2016) and bone loss (MengYong et al., 2008) in animals.Until now the role of DDP in the differentiation of BMSCs into adipogenic cells has not been reported.This study investigated the effect of DDP on the adipogenic differentiation of rat BMSCs and the expressions of adipogenic differentiation related gene peroxisome proliferator-activated receptor gamma (PPARG), lipoprotein lipase (LPL) and fatty acid binding protein 4 (FABP4).The objective was to provide a theory basis for application of DDP to treating the osteoporosis.extracted using hot water, and purified by repeated ethanol precipitation.The Sevage method was to remove the protein, and the phenol-sulfuric acid method was used to determine the DDP content of extract.Finally the content of DDP in extract was 85% m/m.

Preparation of BMSCs
SD rat was killed by cervical dislocation, followed by soaking in 75% ethanol for 10-15 min.The femur and tibia were taken under aseptic condition.After removing the muscle tissue, the bones were washed with PBS for 3 times.The metaphysise was removed, and the bone marrow cavity was exposed.5 mL a-MEM was injected to bone marrow cavity to flush out the bone marrow.The process was repeated for several times.The bone marrow cell suspension was collected in 15 mL centrifuge tubes, and the fat and periosteum were removed by centrifugation at 1500 r/min for 5 min.The cells were suspended with a-MEM, and then were inoculated to the culture dish.After 72 h the culture medium was renewed, followed by renewing once every 2 days.When cell confluence was 80%-90%, the cells are digested with 0.25% trypsin (including 0.02% EDTA), followed by passage for 3 generations.

Detection of cell viability
BMSCs were inoculated to 96-well plate, with the density of 5×10 3 cells/well.After 24 h of culture, the culture medium was exchanged with a-MEM containing 0 (control), 50, 100, 200, 400, 800 μg/mL DDP, respectively, 6 wells for each concentration.After culture for 24, 48 and 72 h, the MTT staining was performed to detect the cell viability (Yang et al., 2014).The optical density (DD) value of each well was determined at 570 nm using Multiskan FC microplate reader (Thermo Fisher Scientific, Inc., MA, USA).

Oil red O staining and quantitative analysis
Dn the 20th day of adipogenic differentiation, the culture medium was removed.The cells were washed with PBS for 3 times, and were fixed with 10% neutral formaldehyde for 30 min, followed by staining with oil red D dye for 30 min.After washing with PBS for 3 times, the cells were observed and photographed under DVM6 optical microscope (Leica Science Lab, Leica Camera AG Berlin, Germany).According to the reported method (Fu et al., 2014), isopropanol was used to extract the oil red D dye, the DD value of cell solution was measured by at 540 nm using microplate.

Detection of mRNA expression of adipogenic differentiation related genes
The mRNA expression of adipogenic differentiation related genes including PPARG, LPL and FABP4 were detected using RT-PCR method.Dn the 20th day of adipogenic differentiation, the cells were collected.The total RNA was extracted with Trizol, followed by reverse transcription to cDNA with Rever Tra Ace  qPCR RT Kit.β-actin was used as internal reference.The primer sequences were as follows: PPARG: sense 5'-TGC TTG TGA AGG ATG CAA GGG T-3' and anti-sense 5'-AGCAAGGCACTTCTGAAACCGA-3'; LPL: sense 5'-TGG ATG GAC GGT GAC AGG AAT GTA-3' and anti-sense 5'-CGG CAG ACA CTG GAT AAT GTT GCT-3'; FABP4: sense 5'-ATC AGC GTA GAA GGG GAC TTG GT-3' and anti-sense 5'-TGG TCG ACT TTC CAT CCC ACT TCT-3' .The PCR amplification conditions were as follows: 95 o C for 5 min; 40 cycles of 50 °C for 2 min, 95 °C for 2 min and 60 o C for 1 min, followed by 72 °C for 1 min.The relative expression level was determined using the 2 −ΔΔCt analysis method (Livak & Schmittgen, 2001).

Statistical analysis
All statistical analysis was carried out using SPSS17.0software (SPSS Inc., Chicago, IL, USA).Data were presented as standard deviation.Comparisons between two groups were performed using t test.P < 0.05 was considered as statistically significant.

Effects of DOP on survival rate of BMSCs
When the concentration of DDP was in 0-800 μg/mL range, there was no significant difference of DD value of cell solution, which represented the survival rate of BMSCs.This indicated that, DDP had no significant toxicity to BMSCs in this concentration range (Figure 1).

Effects of DOP on adipogenic differentiation of BMSCs
Dil red D staining assay showed that, 0, 50 and 100 μg/mL DDP had no significant difference on the DD value of cell solution.When the concentration of DDP was 200, 400 and 800 μg/mL, the DD value was significantly decreased compared with 0, 50 and 100 μg/mL DDP groups (P < 0.05 or P < 0.01).There was no significant difference between 400 and 800 μg/mL DDP groups (P > 0.05) (Figure 2). Figure 3 showed that, the numbers of stained lipid droplets in 200, 400 and 800 μg/mL DDP groups were obviously less than 0, 50 and 100 μg/mL DDP groups, especially for the 800 μg/mL DDP group.This indicated that, DDP with concentration of 200-800 μg/mL could obviously inhibit the adipogenic differentiation of BMSCs.

Discussion
Dsteoporosis is a systemic bone disease characterized by bone mass decrease and bone microstructure degeneration.In this disease, the bone fragility increases, leading to occurrence of fracture (Sandhu & Hampson, 2011).With the improvement of living standards and aging aggravation in more and more countries, the harm caused by osteoporosis is becoming more and more serious.However, the pathogenesis of osteoporosis is  still not clear.In recent years, it is found that there is an important relationship between the osteogenesis-adipogenesis imbalance of BMSCs and the occurrence of osteoporosis.The decrease of bone mass is often accompanied with the increase of the bone marrow adipose tissue.Dnce too much BMSCs differentiate into the adipogenic cells, the number of BMSCs differentiating into osteoblasts will be reduced accordingly (Ye et al., 2014).This has provided a new direction for preventing and treatment of osteoporosis by promoting BMSCs differentiating into osteoblasts and inhibiting the differentiation into adipogenic cells.
DDP is one of the main components of Dendrobium officinale polysaccharide, and also the secondary metabolite.The content and quality of DDP are considered to the key indicators for evaluating the quality of Dendrobium officinale (Jin et al., 2016).DDP is the multi-hydroxyl polymer composed of glucose, mannose, galactose, glucose acid, glucose acid, xylose, Arabia sugar, galactose acid and other monosaccharide (Xia et al., 2012).This study observed the effect of DDP on the adipogenic differentiation of BMSCs.Result showed, DDP had no significant toxicity to BMSCs, and that with concentration of 200-800 μg/mL could obviously inhibit the adipogenic differentiation of BMSCs.
PPARG belongs to the peroxisome proliferator-activated receptors family, and is a key transcription factor for the activation and transcription of the adipogenic differentiation.It is highly expressed in adipose tissue.It is the major regulatory factor in the process of adipogenic differentiation, and is a major regulator of adipocyte related genes expression (Jääger & Neuman, 2011).LPL can bind the protein polysaccharide of extracellular matrix, thus inhibit the osteogenic differentiation and mineralization, and promote the adipogenic differentiation (Linehan et al., 2012).FABP4 belongs to the fatty acid binding protein family.It is a marker gene of adipocyte differentiation and maturation, and is widely involved in the transmembrane transport and metabolism of fatty acids (Lee et al., 2014).Results of this study showed that, after treatment with DDP, the expressions of PPARG, LPL and FABP4 mRNA decreased.This indicates that,

Figure 3 .
Figure 3. Results of oil red D staining assay with different DDP concentrations.DDP, Dendrobium officinale polysaccharide.