| Part I The effect and relative mechanism of deferoxamine on bone mass inovariectomized mice companied with iron accumulationObjective: This study aimed to observe the effect of iron chelator deferoxamine mesylate(DFO) on ovariectomized mice(animal model of type I primary osteoporosis) plus iron intervention, and to explore new therapeutic approach for type I primary osteoporosis.Methods: 8-weeks old C57 BL / 6J female mice were underwent oophorectomy and randomly divided into ovariectomized(OVX) group, ovariectomized + iron intervention group(OVX + Fe group) and ovariectomized + iron + deferoxamine group(OVX + Fe + DFO group). After two weeks, the OVX + Fe group mice and the OVX + Fe + DFO group mice were treated with 0.12 g / kg / w ferric ammonium citrate intervention four weeks via intraperitoneal injection three times a week, while the OVX group mice were treated with saline via the same methods and frequency. Afterwards, the OVX + Fe + DFO group mice were administrated with 4000 mg / Kg / W of DFO for two weeks via intraperitoneal injection twice a day, while mice in the other two groups administrated with saline in the same manner and frequency. To label the active bone formation sites in the mice, we used double labeling, with calcein as a marker. Mice were sacrificed at the end of the administration. The serum ferritin, osteoprotegerin(OPG) or receptor ativator for nuclear factor-! B ligand(RANKL) level were detected by enzyme-linked immuno sorbent assay. Distal femur three-dimensional analysis was detected by micro computed tomography(Micro-CT). The HE staing was conducted to observe the micro architecture of femur. The prussian blue staining was conducted to observe bone iron deposition in femur. Real-time polymerase chain reaction(PCR) was conducted to analyze the expression of OPG and RANKL in bone tissue.Results: The serum ferritin in OVX + Fe group(408.90±17.20μg/ml) was significantly higher than the other groups(OVX group 87.53±3.73μg/ml and OVX+Fe+DFO group 162.30±10.58μg/ml, respectively)(P <0.05). Besides, prussian blue staining showed that the iron deposition in bone of OVX + Fe group mice was the most, and in bone of OVX group mice was the least. These results suggested that iron accumulation could increase systemic and local bone tissue iron accumulation, while DFO could reduce the iron accumulation no matter in serum and in local bone tissue. The bone mass in the OVX + Fe group(0.09±0.02mg/mm3) was significantly lower than the OVX groups(0.13±0.01mg/mm3)(P <0.05). However, after administrated with DFO, the bone mass in the OVX + Fe + DFO group(0.12±0.01mg/mm3) was significantly increased compared with OVX + Fe group(0.09±0.02mg/mm3)(P <0.05). Calcein labeling showed that mineral apposition rate(MAR) in OVX+Fe group was inferior to OVX group and OVX+Fe+DFO group(p<0.05). Compared to OVX group, MAR in OVX+Fe+DFO group decreased(p<0.05). The bone tissue OPG expression in OVX + Fe group(0.75±0.15)was down regulated when compared with the OVX group(1±0.2)(P <0.05). But after administrated with DFO(0.89±0.15), the bone tissue OPG expression was increased significantly compared with the OVX + Fe group(0.75±0.15)(P <0.05). The bone tissue RANKL expression and RANKL/OPG ratio in OVX + Fe group(2.2±0.12 and 2.8±0.4, respectively) was up regulated when compared with the OVX group(1±0.13 and 1±0.5, respectively)(P <0.05). But after administrated with DFO, the bone tissue RANKL expression(1.3±0.2) and RANKL/OPG ratio(1.4±0.3) was decreased significantly compared with the OVX + Fe group(2.2±0.12 and 2.8±0.4, respectively)(P <0.05). The levels of OPG and RANKL in serum were as the same as the results of real-time PCR.Conclusion: Iron accumulation decreased bone mass in OVX mice through affecting the RANKL/receptor ativator for nuclear factor-! B(RANK)/OPG axis, while DFO might reduce iron accumulation and partially restored the bone mass in animal model of type I primary osteoporosis.Part II The effect and related mechanism of DFO on FAC treated osteoblast physiologyObjective: This study aimed to observe the effect of DFO on FAC treated osteoblast physiology, and to explore the relative mechanism.Methods: Primary osteoblasts were collected from the calvaria of C57 BL / 6J newborn mice and were subjected to osteoblast mineralization culture. After alkaline phosphatase(ALP) staining to confirm the purity of OB, we passaged the cells to the third generation. CCK8 was conducted to explore the concentration of FAC or DFO treatment. Then we divided the OBs into four groups including the control group(ctrl group), 50 u M/L FAC treated group(Fe group), the 50 u M/L Fe plus 20 u M/L DFO treated group(Fe+DFO group) and 50 u M/L Fe plus 20 u M/L DFO and 500ng/ml DKK-1 group(Fe+DFO+DKK-1 group). Besides, we add serial concentration of DFO(0, 5, 10, 20 u M/L) in the Fe group OBs respectively. Afterwards, ALP staining and ALP activity assay were conducted to detect the osteoblast activity. Bone formation in vitro was performed then confirmed by alizarin red staining. Expression of Runx2, Sp7, Bglap, Axin2, OPG and Sod1 were measured by real-time PCR. DCFH fluorescent probe was applied to analyze the level of reactive oxygen species(ROS). Westernblot was conducted to analyze the protein level of Axin2 and OPG.Results: CCK8 showed that treated with 50 u M/L Fe or 50 u M/L Fe plus 20 u M/L DFO did not affect the cell viability. When compared with the ctrl group or the Fe+DFO group, the expression of Runx2, Sp7, Bglap, Axin2 and OPG were down-regulated in the Fe group, while the expression of Sod1 was up-regulated. ALP staining and activity assay as well as the calcium nodules in the Fe group was inferior to the ctrl group or the Fe+DFO group. The level of ROS in the Fe group was superior to the ctrl group or the Fe+DFO group. Treated with serial concentration of DFO plus Fe 50 u M/L increased the calcium nodules and Axin2 protein level, while decreased the ROS level in a dose dependent manner. There is no significant difference in ROS level between the Fe+DFO+DKK-1 group and the Fe+DFO group. However, the protein levels of OPG and Axin2 as well as the calcium nodules in the Fe+DFO+DKK-1 group were inferior to the Fe+DFO group.Conclusion:FAC might through Wnt signaling pathway to up-regulates ROS level, decrease osteogenetic related genes expression in OB and inhibit OB physiology. DFO decreases FAC induced ROS up-regulation, and restore the inhibition of FAC on OB physiology partly. |
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