ABSTRACT
A water-soluble polysaccharide (SPAW) was purified from Safflower and it was identified to be (1â3)-linked ß-d-Glucan. The therapeutic effect and underlying mechanism of SPAW on steroid-induced avascular necrosis of the femoral head (SANFH) in a rabbit model was performed here. The abnormal histopathologic changes and apoptosis of femoral head in model group were significantly reverted after SPAW (25, 100 and 200â¯mg/kg) administration for 60 days, as evidenced by the a decline of empty lacunae rate, the average bone marrow fat cell size and the proportion of apoptotic cells. Furthermore, administration of SPAW significantly decreased the Bax and caspase-3 protein expression, but increased the protein expression of Bcl-2 when compared these in model rabbits. Meanwhile, increased hydroxyproline (HOP) and decreased serum hexosamine (HOM) concentration in rabbit serum were turned to the opposite way. The present study suggested that SPAW may provide an alternative treatment for the treatment of SANFH.
Subject(s)
Carthamus tinctorius/chemistry , Femur Head Necrosis/drug therapy , Glucans/therapeutic use , Animals , Apoptosis/drug effects , Caspase 3/metabolism , Female , Femur Head/pathology , Femur Head Necrosis/chemically induced , Glucans/chemistry , Glucans/isolation & purification , Hexosamines/metabolism , Horses , Hydroxyproline/metabolism , Male , Methylprednisolone Hemisuccinate , Molecular Weight , Proto-Oncogene Proteins c-bcl-2/metabolism , Rabbits , Rats , bcl-2-Associated X Protein/metabolismABSTRACT
A water-soluble polysaccharide (APP-AW) was isolated from Agrimonia pilosa and prepared to three sulphated derivatives (S1, S2 and S3). The results showed that pre-treatment with APP-AW, S1, S2 and S3 each at the concentration of 50 µg/mL for 48 hours was able to prevent cytotoxicity induced by 1 µmol/L dexamethasone (Dex) in MC3T3-E1 cells via inhibition of apoptosis, which is in line with the findings in flow cytometry analysis. Meanwhile, the decreased ALP activity, collagen content, mineralization, BMP2, Runx2, OSX and OCN protein expression in DEX-treated MC3T3-E1 cells were reversed by the addition of APP-AW, S1, S2 and S3. Moreover, APP-AW, S1, S2 and S3 rescued DEX-induced increase of Bax, cytochrome c and caspase-3 and decrease of Bcl-2, Wnt3, ß-catenin and c-Myc protein expression in MC3T3-E1 cells. Our findings suggest that pre-treatment with APP-AW, S1, S2 and S3 could significantly protect MC3T3-E1 cells against Dex-induced cell injury via inhibiting apoptosis and activating Wnt/ß-Catenin signalling pathway, thus application of these polysaccharides may be a promising alternative strategy for steroid-induced avascular necrosis of the femoral head (SANFH) therapy.
Subject(s)
Agrimonia/chemistry , Dexamethasone/pharmacology , Polysaccharides/pharmacology , Protective Agents/pharmacology , Signal Transduction/drug effects , Wnt Signaling Pathway/drug effects , beta Catenin/metabolism , 3T3 Cells , Animals , Apoptosis/drug effects , Femur Head Necrosis/drug therapy , MiceABSTRACT
The present study showed that low level of miR-107 in femoral head tissues from rats suffering steroid-induced osteonecrosis of the femoral head (SANFH) was associated with high degree of osteonecrosis and apoptosis. In vitro assay, pretreatment with an Agrimonia pilos polysaccharide (APP-AW) and its sulphated derivatives (S2) at 50 µg/ml for 48 h or overexpressing miR-107 were able to prevent cytotoxicity induced by 1 µM dexamethasone (DEX) in MC3T3-E1 cells via inhibition of apoptosis. Meanwhile, the decreased ALP activity, collagen content, BMP2, Runx2, OSX and OCN protein expression in DEX-treated MC3T3-E1 cells were rescued by the addition of APP-AW and S2, or miR-107 transfection. Moreover, DEX-induced increase of Bax, cytochrome c and caspase-3, as well as decrease of Bcl-2, Wnt3, ß-catenin and c-Myc protein expression in MC3T3-E1 cells were also reversed. These findings suggest that APP-AW and S2 promote cell proliferation and osteogenic differentiation by enhancing miR-107 during the development of SANFH.
Subject(s)
Agrimonia/chemistry , Cell Differentiation/drug effects , MicroRNAs/genetics , Osteogenesis/drug effects , Polysaccharides/chemistry , Polysaccharides/pharmacology , Sulfates/chemistry , Animals , Apoptosis/genetics , Biomarkers , Cell Line , Cell Proliferation/drug effects , Male , MiceABSTRACT
In this study, one homogeneous polysaccharide (APP-AW), with an average molecular weight of 9550 Da, was purified from Agrimonia pilosa. Analysis by gas chromatography (GC), methylation, UV, Infrared spectra (IR), 1D and 2D nuclear magnetic resonance (NMR) spectroscopy indicated that APP-AW was a ß-(1 â 3)-d-glucan. The effect of APP-AW on dexamethasone (Dex)-induced apoptosis in osteoblasts was also examined. Pretreatment of APP-AW (100 µg/ml) significantly attenuated cell loss and apoptosis induced by Dex (1 µM) in osteoblasts as determined by MTT, Annexin V-FITC/ propidium iodide (PI) and Transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining assay. Collectively, the present study demonstrated that APP-AW might be an alternative therapeutics for the treatment of SANFH via reducing Dexinduced bone cellular apoptosis.
Subject(s)
Agrimonia/chemistry , Glucans/pharmacology , Osteoblasts/drug effects , Plant Components, Aerial/chemistry , Animals , Apoptosis/drug effects , Dexamethasone , Femur Head Necrosis/chemically induced , Femur Head Necrosis/drug therapy , Glucans/chemistry , Glucans/isolation & purification , Molecular Weight , RatsABSTRACT
A water-soluble polysaccharide (SPS) was purified from dried safflower (Carthamus tinctorius L.) and its structure was identified using a combination of chemical and instrumental analysis. SPS has a repeating backbone of 1,4,6-ß-Glcp, which was attached with T-ß-Glcp at its C6 position along the main chain in the molar ratio of 1:1. A steroid-induced avascular necrosis of the femoral head (SANFH) model was established in mice injected with dexamethasone (50â¯mg/kg) twice per week for 6â¯weeks. Following SPS treatment at 25 and 100â¯mg/kg for 60â¯days, the decreased bone mineral density, abnormal histopathological changes, the increased rate of empty lacunae and apoptosis rate of osteocytes of femoral head in mice induced by dexamethasone was significantly reversed. Meanwhile, increased serum hydroxyproline (HOP) and decreased serum hexosamine (HOM) concentration in mice were turned to the opposite trend with increasing dosage of SPS, thus leading to a high rate of HOM/HOP. In conclusion, SPS may serve as a potential agent for the treatment of SANFH.
Subject(s)
Carthamus tinctorius/chemistry , Femur Head Necrosis/drug therapy , Polysaccharides/administration & dosage , Animals , Apoptosis/drug effects , Caspase 3/genetics , Disease Models, Animal , Femur Head Necrosis/blood , Femur Head Necrosis/chemically induced , Femur Head Necrosis/physiopathology , Hexosamines/blood , Humans , Hydroxyproline/blood , Mice , Osteocytes/drug effects , Osteocytes/pathology , Polysaccharides/chemistry , Rats , Steroids/toxicityABSTRACT
The present study aimed to investigate the effects of a purified polysaccharide (SPS) from the safflower in a cellular model of steroid-associated necrosis of the femoral head (SANFH), which was established in primary murine osteoblasts suffering dexamethasone pretreatment. After treatment with SPS (25, 50 and 100⯵g/ml), the degree of necrosis induced by dexamethasone was significantly reduced in osteoblasts as evidenced by an increase of cell viability and a decrease of apoptosis in osteoblasts. Furthermore, pretreatment with SPS (25, 50 and 100⯵g/ml) significantly attenuated the activation of caspase-3 and cleavage of PARP relative to the model control cells. The addition of caspase-3 inhibitor (Z-DEVD-FMK) in dexamethasone-treated osteoblasts resulted in the inefficiency of SPS for inhibiting cellular apoptosis. Dose-dependent increases in alkaline phosphatase (ALP) activity, collagen synthesis and mineralization were also observed in SPS-treated osteoblasts at 72â¯h. The present study demonstrates that SPS may alleviate dexamethasone associated osteonecrosis by inhibiting caspsae-3-mediated apoptosis and may provide an alternative treatment for SANFH.
Subject(s)
Apoptosis/drug effects , Carthamus tinctorius/metabolism , Caspase 3/metabolism , Femur Head Necrosis/drug therapy , Polysaccharides/pharmacology , Animals , Apoptosis Regulatory Proteins/metabolism , Cell Survival/drug effects , Dexamethasone/pharmacology , Femur Head Necrosis/chemically induced , Femur Head Necrosis/metabolism , Mice , Osteoblasts/drug effects , Rats , Rats, Wistar , Signal Transduction/drug effects , Steroids/pharmacologyABSTRACT
Steroid-induced avascular necrosis of the femoral head (SANFH) is a major limitation of long-term or excessive clinical administration of glucocorticoids. Fludarabine, which is a compound used to treat various hematological malignancies, such as chronic lymphocytic leukemia, acts by down-regulating signal transducer and activator of transcription 1 (STAT1) by inhibiting STAT1 phosphorylation in both normal and cancer cells. This study assessed the effects of fludarabine in vitro (primary murine osteoblasts) and in vivo (rat SANFH model). In vitro, pretreatment with fludarabine significantly inhibited Dexamethasone (Dex)-induced apoptosis in osteoblasts, which was examined by TUNEL staining. Treatment with Dex caused a remarkable decrease in the expression of Bcl-2; an increase in cytochrome c release; activation of BAX, caspase-9, and caspase-3; and an obvious enhancement in STAT1 phosphorylation. However, treatment resulted in the up-regulation of caspase-3 expression. Enhanced P-STAT1 activity and up-regulation of caspase-3 expression were also observed in osteoblasts. In vivo, the subchondral trabeculae in fludarabine-treated rats exhibited less bone loss and a lower ratio of empty lacunae. Taken together, our results suggest that STAT1-mediated up-regulation of caspase-3 is involved in osteoblast apoptosis induced by Dex and indicates that fludarabine may serve as a potential agent for the treatment of SANFH.