| BackgroundThe microgravity environment experienced by human crewmembers during space flight or patients during long-term bed rest has an immediate impact on many of the body’s biological systems. One of the most significant effects as a result of exposure to microgravity is bone loss, which leads to an increase in fracture risk. Long-term exposure to a microgravity environment leads to enhanced bone resorption in the early phase and then a sustained reduction in bone formation for the duration of weightlessness. Knowledge of microgravity-induced bone loss and the development of anti-catabolic strategies that retard bone loss are important and challenging for the care of the geriatric population and astronauts.Up to date, the underlying mechanism of bone loss induced by microgravity is unclear yet. Various countermeasures fail to prevent the decrement in bone mass and formation caused by unloading, however, no ideal treatments have been found. Over the past years, the role of ROS in the microgravity-induced bone loss has been drawn great attention. It has been widely accepted that ROS plays a key role in both the postmenopausal and senile osteoporosis. Up to date, some treatments aiming at antioxidant stress have been developed and good results were achieved in the clinical applications. Therefore, it is extremely worth investigating the role of ROS in the microgravity-induced bone loss.Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a phenolic natural product isolated from the rhizome of Curcuma longa (turmeric). It has been widely used in Eastern populations, particularly as a traditional medicine in India and China, for the treatment of many diseases, including diabetes and diseases of hepato-biliary, skin, rheumatoid, and gastrointestinal system. Recently, curcumin has caught scientific attention as a potential therapeutic agent in ovariectomized mature rodent model of postmenopausal osteoporosis and experimental periodontitis bone loss model. In addition, treatment with curcumin improved bone microarchitecture and enhanced mineral density in APP/PS 1 transgenic mice, a model of alzheimer’s disease. Furthermore, it has been reported that curcumin is a bifunctional antioxidant because of its ability to react directly with reactive species and to induce an up-regulation of various cytoprotective and antioxidant proteins. In addition, curcumin is able to bind vitamin D receptor (VDR), and activate transcription of a VDR-target gene.In the present study, we explored whether long-term treatment with curcumin had beneficial effect in rats exposed to HLS in vivo and its underlying mechanism. In addition, we explored the effect of incubation with curcumin on osteoblastic and osteoclastic function in MC3T3-E1 cells and RAW264.7 cells cultured in rotary wall vessel bioreactor.Objectives1. To study the functional changes of both metabolism and differentiation of incubation with curcumin in MC3T3-E1 cells cultured in rotary wall vessel bioreactor, and explore the underlying mechinism of these biological effects.2. To study the functional changes of both metabolism and differentiation of incubation with curcumin in RAW264.7 cells cultured in rotary wall vessel bioreactor, and explore the underlying mechinism of these biological effects.3. To investigate the changes of bone metabolism in rats exposed to modeled microgravity in vivo, and explore its underlying mechanism.Methods1. Rotary wall vessel bioreactor (RWVB) was used to model microgravity in vitro. The MC3T3-E1 cells were exposed to modeled microgravity and treated with curcumin (4μM) for 96 hours. The intracellular ROS, OPG and RANKL content in supernatant were determined. ALP activity was determined after 7 days of treatment with curcumin (4μM). The mRNA levels were determined by quantitative real time chain reaction(qRT-PCR) and protein expression of VDR were determined using Western blot.2.The MC3T3-E1 cells were exposed to modeled microgravity and treated with curcumin (4 μM) or 1,25-dihydroxyvitamin D (1,25D; 10 nM) or curcumin+1,25D. The intracellular ROS, OPG, and RANKL content in supernatant were determined.3. The RAW264.7 cells were exposed to modeled microgravity and stimulated with RANKL (20 ng/ml), being treated with curcumin (4μM) for 96 hours. Then, ROS formation, CathK and TRAP mRNA expression were determined. After the cells were TRAP-stained, multinucleated TRAP-positive cells with different numbers of nuclei were counted in two different categories:3 to 30 nuclei and those with greater than 30 nuclei.4. Hind-limb suspension (HLS) was used to model microgravity in vivo. SD rats were divided into 4 groups of 12 animals each (1) control rats treated with vehicle (Con); (2) HLS rats treated with vehicle; (3) control rats treated with curcumin (Con+CUR); (4) hind-limb-suspended rats treated with curcumin (HLS+CUR). SD rats were suspended for a total of 6 weeks. At the end of the experimental period, all rats were fasted for 12 hours, and then blood sample was collected from their abdominal aorta.1,25-(OH)2D3 levels were measured from serum by enzyme immunoassay. MDA levels of plasma were determinated by using a kit.5. After 6 weeks of experimental period, urinary DPD excretion was quantified by using an EIA kit and the data were corrected for the urinary creatinine levels. Creatinine (CRE) levels were determined with QuantiChrom Creatinine Assay Kits.6. After 6 weeks of tail-suspension, the femurs were excised from each rat and bone homogenates were prepared. Bone homogenates were used for the determination of MDA by using a kit. t-SH of bone homogenates was determined by using Glutathione Assay Kits. mRNA expression level of VDR、TRAP、CN、RANKL、 OPG in bone homogenates of SD rats were determined by quantitative real time polymerase chain reaction(qRT-PCR). VDR levels in the bone homogenate of SD rats were determined by Western blot.7. Bone mineral density (BMD) were measured ex vivo with a dual-energy X-ray absorptiometry NORLAND XR-46 (Norland Co. Fort Atkinson, WI, USA) using the small animal program set to a high-resolution mode. Samples were placed on an acrylic platform of uniform 38.1-mm thickness. The BMD of whole tibiae was obtained. The coefficient of variation (CV) was 1% for BMD.8. After 6 weeks of tail-suspension, histomorphometric data were collected with the Bioquant Bone Morphometry System. Osteoclast surface (Oc.S/BS) and osteoblast surface (Ob.S/BS) were obtained. Measurements were performed at the metaphyseal region of the proximal tibiae, Eight bone sections were analyzed per animal.9. Trabecular bone morphometry within the metaphyseal region of proximal tibiae was quantified using micro-CT. Trabecular morphometry was characterized by measuring trabecular bone volume fraction (BV/TV), trabecular thickness (Tb. Th), trabecular separation (Tb.Sp) and trabecular number (Tb. N.).10. The mechanical strength including ultimate compressive load (newton), the stiffness (newton per millimeter), and the energy (megajoule) of the left femur were measured using a compression test and a three-point bending test using a mechanical strength analyzer.Results1. Compared with the control group, MC3T3-E1 cells cultured in RWVB showed increased ROS formation (p=0.002). Incubation with curcumin suppressed ROS formation (p=0.014)2. Compared with the control group, exposure to modeled microgravity of MC3T3-E1 cells led to an augmentation of ratio of RANKL/OPG (p<0.001), which was decreased by treatment with curcumin (p<0.001).3. MC3T3-E1 cells cultured in RWVB showed reduced mRNA levels (p<0.001) and protein expression) of VDR. Treatment with curcumin enhanced VDR mRNA levels (p<0.001) and upregulated VDR protein expression (p<0.001) in MC3T3-E1 cells cultured in RWVB.4. Compared with the control group, MC3T3-E1 cells cultured in RWVB showed reduced osteoblastic differentiation marked by decreased ALP activity(p=0.005). Incubation with curcumin enhanced osteoblastic differentiation (p=0.020).5. Compared with the Con group, MC3T3-E1 cells cultured in RWVB (MG group)showed increased intracellular ROS formation (p=0.001); As compared to the MG group, ROS formation was suppressed significantly in MG+curcumin (4 μM) group and MG+ curcumin (4 μM)+1,25D(10nM) group (p=0.006;p=0.001) No significance was found betweenMG group and MG+1,25D group (p=0.54); There was also no significance between MG+CUR group and MG+1,25D+CUR group (p=0.74)6. Compared with the Con group, MG group led to an augmentation of the ratio of RANKL/OPG (p=0.007); As compared to the MG group, the ratio of RANKL/OPG reduced significantly (p<0.001) in MG+curcumin group, MG+ 1,25Dgroup and MG+curcumin+1,25D group (p=0.01; p=0.009; p=0.005).No significance was found between MG+CUR group and MG+1,25D group (p=0.55); There was also no significance between MG+CUR group and MG+1,25D+CUR group (p=0.44)7. Compared with the control group, MC3T3-E1 cells cultured in RWVB showed reduced osteoblastic differentiation marked by decreased ALP activity (p=0.04) Incubation with curcumin (4μM) and 1,25D (10 nM) or curcumin (4 μM)+1,25D (10 nM) enhanced osteoblastic differentiation (p=0.07; p=0.009; p=0.13) No significance was found between MG+CUR group and MG+1,25D group(p=0.85) in ALP activity; There was also no significance between MG+CUR group and MG+1,25D+CUR group (p=0.54)8. RAW254.7 cells cultured in RWVB showed increased ROS formation (p<0.001). Treatment with curcumin reduced ROS levels(P<0.05).9. RAW254.7 cells cultured in RWVB enhanced osteoclastic differentiation marked by increased CathK (p<0.001) mRNA levels, and increased osteoclastogenesis marked by increased TRAP mRNA levels (p<0.001). Treatment with curcumin suppressed osteoclastic differentiation and osteoclastogenesis marked by reduced mRNA levels of CathK (p<0.001) and TRAP (p<0.001).10. RAW254.7 cells cultured in RWVB increased the number of TRAP-positive multinucleated osteoclasts (3<nuclei<30, p<0.001; nuclei>30, p<0.001). Treatment with curcumin reduced the number of TRAP-positive multinucleated osteoclasts (3<nuclei<30, p=0.001; nuclei>30, p<0.001).11. SD rats were exposed to HLS for 6 weeks and treated with or without curcumin., the soleus muscle-to-body mass ratios were significantly lower (pO.OOl) in rats exposed to HLS, which confirmed the efficacy of simulated microgravity in this set of experiments. HLS led to a significant reduction in body weight (p<0.001) and food intake (p=0.010), and curcumin treatment had significant effect on body weight and food intake.12. When compared with the control rats, BMD of the whole tibia (p=0.011) were lower in HLS rats; When compared with HLS rats, BMD of the whole tibia were higher in HLS rats treated with curcumin(p=0.032).13. When compared with the control rats, Tb.Th (p=0.002), Tb.N (p<0.001), and BV/TV (p<0.001) of proximal tibiae were lower and Tb.Sp (p<0.001) were higher in HLS rats. When compared with HLS rats, Tb.Th(p=0.013), Tb.N (p=0.018), and BV/TV(p=0.008) of the proximal tibiae were higher, and Tb.Sp (p<0.001) of the proximal tibiae were lower in HLS rats treated with curcumin.14. When compared with the control rats, Ob.S/BS (p<0.001) of proximal tibiae were lower and Oc.S/BS (p<0.001) of proximal tibiae were higher in HLS rats. When compared with HLS rats, Ob.S/BS of the proximal tibiae were higher and Oc.S/BS were lower in HLS rats treated with curcumin.15. When compared with the control rats, ultimate load (p=0.007), stiffness (p<0.001), and energy (p=0.004) of femoral diaphysis were lower, When compared with HLS rats, ultimate load(p=0.027), stiffness (p=0.015), and energy (p=0.021) of femoral diaphysis were higher in HLS rats treated with curcumin.16. Compared with the control rats, mRNA levels of TRAP (p<0.001) and mRNA ratio of RANKL-to-OPG (p<0.001) of distal femurs were higher in HLS rats, and mRNA levels of osteocalcin (p<0.001) of distal femurs was lower in HLS rats.In HLS+curcumin group, mRNA levels of TRAP(p<0.001) and mRNA ratio of RANKL-to-OPG of distal femurs (p<0.001) were lower, and mRNA levels of osteocalcin of distal femurs (p<0.001) was higher than that in the HLS group.17. Exposure to HLS led to reduction of the levels of mRNA (p<0.001) and protein expression (p<0.001)of VDR in femurs. Treatment with curcumin enhanced VDR mRNA levels (p<0.001) and upregulated VDR protein expression (p<0.001) in rats exposed to HLS.18. Exposure to HLS led to reduction of serum levels of 1,25-(OH)2D3 (p<0.001). Treatment with curcumin did not affect serum levels of 1,25-(OH)2D3 (P>0.05)19. Compared with the control rats, urinary DPD excretion(p<0.001) were higher in HLS rats, and mRNA levels of osteocalcin (p<0.001) of distal femurs was lower in HLS rats. In HLS+curcumin group, urinary DPD excretion (p=0.003) were lower than that in the HLS group.20. Compared with the control rats, t-SH levels (p=0.002) of distal femurs were lower in the HLS group. Treatment with curcumin enhanced the levels of t-SH in distal femurs (p<0.001).21. Compared with the control rats, MDA levels of plasma (p=0.003) and distal femurs (p=0.006) were higher; Treatment with curcumin reduced the levels of MDA in plasma (p=0.011) and distal femurs (p=0.017).Conclusions1. Curcumin inhibited modeled microgravity-induced reactive oxygen species (ROS) formation and enhanced osteoblastic differentiation in MC3T3-E1 cells cultured in RWVB. Curcumin upregulated vitamin D receptor (VDR) expression in MC3T3-E1 cells exposed to modeled microgravity.2. In cultured RAW264.7 cells, curcumin reduced modeled microgravity-induced ROS formation and attenuated osteoclastogenesis in RAW264.7 cells exposed to modeled microgravity.3. Curcumin alleviated HLS-induced bone loss in rats, possibly via suppressing oxidative stress and upregulating VDR expression. |
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