Curcumin reduces the cytotoxicity and osteogenesis inhibition induced by oxidative stress via inhibited the NF-κB and MAPK signaling

Excessive oxidative stress is considered a significant pathogenic factor leading to the failure of bony fusion at the interface between implanted materials and the host. Curcumin has demonstrated anti-inflammatory and antioxidant properties. However, there were few studies investigating the effects of curcumin on osteogenesis, as well as the underlying mechanism involved in pre-osteoblastic MC3T3-E1 cells. We established an oxidative damage model by exposing MC3T3-E1 cells to hydrogen peroxide (H2O2) for 24 h before osteogenic induction with curcumin. We discovered that oxidative stress diminished the viability of MC3T3-E1 cells, inhibited alkaline phosphatase (ALP) activity and calcium mineralization, and down-regulated osteogenesis-related gene expression; low concentrations of curcumin (1–10 μM) reversed the effects of oxidative stress during osteogenic induction, but high concentrations (20 μM) did not. Notably, curcumin exerted its protective effects in part by inhibiting the phosphorylation of JNK and p38 induced by H2O2 and by blocking of the NF-κB signaling pathway. In vivo analysis indicated that curcumin alleviated the deterioration of the micro-architecture of trabecular bone and the reduction in bone mineral density (BMD) of the distal femur; the protective effects of curcumin were dose-dependent. Taken together, these results indicate that curcumin can exert protective effects on osteogenesis and bone remodeling via inhibition of the NF-κB and MAPK pathways. This study suggests that curcumin might be effective in preventing oxidative stress and facilitate osteogenesis.


INTRODUCTION
The clinical demands for bone repair materials are enormous in spinal fusion.To date, many kinds of bone repair materials have achieved good bony fusion in spine surgery.However, several patients still suffer intervertebral bony fusion failure, with an incidence of approximately 4-20% [1,2].Our previous studies have found that fibrosis at the interface between bone and repair materials were main pathology in the bone nonunion case of anterior lumbar interbody fusion (ALIF) porcine model [3,4].Although excellent materials and advanced technology have developed rapidly, promoting bone formation and avoiding fibrosis remains a large challenge for researchers.The key regulatory mechanism controlling interactions between bone repair materials and the host during bone formation is currently unclear.To elucidate the molecular mechanisms of spinal fusion in anterior Research Paper lumbar interbody fusion, we examined gene-expression profiles after the implantation of bone repair materials using microarray technology, and the data analysis showed that mesenchymal stem cells (MSCs) around bone repair materials primarily undergo oxidative stress, hypoxic stress and endoplasmic reticular stress in the early stages [5,6].Oxidative stress modulates bone formation and the recruitment, proliferation and osteogenic differentiation of MSCs; oxidative stress might therefore be an important factor in regulating osteogenesis homeostasis.
As a critical mediator of pathophysiological responses, oxidative stress describes an imbalance between excess production of reactive oxygen species (ROS), including oxygen free radicals, and insufficient antioxidant capacity [7,8].The inhibition of bone remodeling by oxidative stress has been extensively studied [9]; ROS can lead to osteoblast apoptosis, as well as a reduction in osteogenesis.Many recent studies have suggested that oxidative stress plays an important role in osteoporosis pathogenesis, in a shift from the predominant "estrogen-centric" view of osteoporosis pathogenesis; the levels of ROS were elevated, and enzymatic antioxidants, such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), were decreased in ovariectomized animals [10,11].
Curcumin (diferuloylmethane), a phenolic antioxidant extracted from curcuma longa and turmeric, has been reported to have anti-cancer, antioxidant, and anti-inflammatory properties [12][13][14].Its negligible toxicity and multiple therapeutic effects have attracted considerable attention in medicine [15].Cytoprotective roles of curcumin have been demonstrated, including the alleviation of tissue damage stemming from lipid peroxidation, free radicals, and hypoxia [16].Additionally, curcumin has demonstrated renal-protective effects by inhibiting oxidative stress in rhabdomyolysisinduced acute kidney injury in rats [17].Curcumin has also demonstrated protective functions against oxidative injury during inflammation or aging-related scenarios [18].A low dose of dietary curcumin significantly decreased inflammation in the brain in a mouse model of Alzheimer's disease [19].Curcumin has been reported to act on multiple cellular targets and signaling pathways to suppress the production of mediators of oxidative damage.
Due to their long-standing use and minimal side effects compared to western medicine, natural product compounds incorporating antioxidant and antiinflammatory properties may be a promising and safe solution to chronic disease [20].The aims of the present study were to investigate the capacity of curcumin to prevent H 2 O 2 -induced oxidative stress and to examine the underlying mechanism.We showed that H 2 O 2induced oxidative stress was mediated by p-P65, p-JNK, and p-P38 activation in MC3T3-E1 cells, and this effect was attenuated with curcumin treatment.In our mouse model, systemic administration of curcumin significantly improved bone mineral density and trabecular bone architecture.This explorative study provides evidence that this natural pleiotropic agent may effectively prevent oxidative stress.

A cellular model of oxidative stress, and curcumin inhibition of H 2 O 2 -induced cytotoxicity
We mimicked oxidative stress conditions after with cells exposed to H 2 O 2 in vitro.We evaluated the level of oxidative stress in different conditions by measuring intracellular ROS levels with flow cytometry.Levels of intracellular ROS were increased in a dose-dependent manner after treatment with H 2 O 2 for 1 h and 24 h.The levels of intracellular ROS significantly increased when exposed to 0.3 mM H 2 O 2 for 1 h (Figure 1A, 1B), which was slightly higher than the levels at 24h.MC3T3-E1 cell viability decreased gradually when MC3T3-E1 cells were incubated with H 2 O 2 for 24 h at concentrations over 0.3 mM and for 48 h at concentrations over 0.2 mM (Figure 1C).Co-culture with curcumin alone at concentrations of 20 μM or lower for 24 h did not affect cell viability, but the viability decreased at a concentration of 20 µM at 48 h (Figure 1D).Importantly, 10 µM or lower curcumin pretreatment in cells exposed to 0.3 mM H 2 O 2 rescued cell viability, but this effect was not seen at the 20µm curcumin concentration (Figure 1E).This result indicates that curcumin can partially protect H 2 O 2 -induced cytotoxicity below 10 µM.

Inhibition of osteogenic differentiation by H 2 O 2 is reversed by curcumin treatment in vitro
ALP activity is considered an early indicator for osteogenesis, ALP activity and staining were measured at 7 days, and calcium levels were measured at 21 days.The ALP activity decreased gradually when the concentration of H 2 O 2 was greater than 0.1 mM, and the calcium level decreased markedly when the concentration was over 0.3 mM.Our experiments focused on the osteogenesis of the cells, so we selected 0.3 mM for this trial.The ALP activity and the calcium level in the cells decreased markedly when co-cultured with 20 µM curcumin alone and we therefore choose 20 µM as the critical curcumin concentration used to determine the curcumin protective effects, For the overall cellular osteogenic effects, we chose the representative concentrations of 1,10,20 µM curcumin in the experiments (Figure 2A, 2B).
The osteogenic medium containing different concentrations of curcumin were added after the cells were exposed to H 2 O 2 for 24 h.Co-treatment of 1 and 10 µM curcumin reversed H 2 O 2 -induced dysfunction, as demonstrated by increased ALP staining, activity and calcium level, but 20 µM curcumin did not reverse the H 2 O 2 effect (Figure 2C-2F).10 µM curcumin treatment reversed H 2 O 2 -induced calcium level dysfunction markedly, and 1 µM treatment reversed the ALP activity dramatically.

Curcumin reversed the effects of oxidative stress on the expression of osteogenesis-related genes
We further investigated the effects of different concentrations (1, 10, 20 µM) of curcumin on the expression of osteogenic genes, including alp, col1a1, runx2 and opn, using RT-PCR.The expression of these markers decreased dramatically in the H 2 O 2 -treated group.The expression levels of three osteogenic genes, alp, runx2 and collagen 1, were significantly upregulated with 1 µM curcumin treatment after exposure to 0.3 mM H 2 O 2 , and the expression of opn was up-regulated in all groups compared with the H 2 O 2 group (Figure 3A-3D).All of the genes except for opn decreased with 20 µM curcumin treatment.Therefore, the above results suggest that 1 and 10 µM curcumin treatment partially protected the inhibition of osteogenic differentiation by oxidative stress through the promotion of osteogenic gene expression.

Expression of RANKL and IL-6 was inhibited by curcumin
Osteoblasts interact with osteoclasts synergistically to regulate the metabolism of bone tissue.The osteoblasts always secrete several cytokines, which affect the activity of the osteoclasts in bone remodeling.RANKL and IL-6 are considered two important factors secreted by osteoblasts.After exposure to 0.3 mM H 2 O 2 for 24 h, the expression levels of RANKL and IL-6 increased over 7 days of osteogenic induction.However, when cells were co-cultured with curcumin, the elevated expression of RANKL and IL-6 was partially inhibited, especially at the 1 μM and 10 μM curcumin (Figure 4A, 4B).

Curcumin reduced the production of reactive oxygen species triggered by H 2 O 2 in MC3T3-E1 cells
We have observed that the production of ROS significantly increased when cells were exposed to H 2 O 2 .The production of ROS increased with increasing concentrations of H 2 O 2 , and the ROS come to the highest level when cells were treated with 0.3 mM H 2 O 2 for 1 h.ROS production was partially suppressed by pretreatment with different concentrations (1, 10, 20 µM) curcumin for 24 h.As shown in the representative photographs of DCF fluorescence, as well as in the relative DCF quantification (Figure 5A, 5B), 20 µM curcumin markedly reduced intracellular ROS levels induced by H 2 O 2 .

NF-κB and MAPK pathways are involved in the anti-oxidative stress effects of curcumin in MC3T3-E1 cells
As a nuclear transcription factor, NF-κB plays a key role in regulating the expression of genes involved in osteogenesis.The expression of the phosphorylated p65 subunit was increased when the cells were exposed to H 2 O 2 .Meanwhile, IκB-α, which is bound to the p65 subunit in the cytoplasm, was diminished in the H 2 O 2treated groups.In contrast, curcumin (1 µM) treatment reduced the expression of p-P65 obviously but the 10, 20 µM failed, conversely, the level of IκB-α in accordance with the expression of p-P65.Therefore, curcuminmediated protection of cells from oxidative stress in osteogenesis, might occur via the inhibition of the NF-κB signaling pathway (Figure 6A-6C).It has been previously reported that MAPK pathways, including ERK1/2, P38 and JNK, are involved in osteogenesis.To elucidate whether curcumin reversed oxidative stress through the regulation of MAPK signaling pathways, we pre-treated MC3T3-E1 cells for 24 h and then cultured the cells with H 2 O 2 for 1 h.Western blot analysis showed that the levels of p-P38 and p-JNK increased and the p-ERK1/2 levels decreased with exposure to H 2 O 2 ; curcumin partially inhibited P38 and JNK phosphorylation, with marked suppression at 20µM, 1µM respectively, the 10 µM concentration had impact on the levels of p-P38 and p-JNK as well.curcumin had opposite effects on ERK1/2 levels (Figure 6D-6G).Here, the data indicated that the H 2 O 2mediated decrease in the osteogenesis of the MC3T3-E1 cells might be associated with the MAPK pathway.Although the most significant suppression of transcription factor with different concentrations of curcumin, curcumin exerted protection might via inhibition of NF-κB and MAPK signaling pathway.

Curcumin in vivo inhibited oxidative stress
Oxidative stress is considered the main pathological mechanism in the ovariectomy (OVX) mouse model, so we used this ovariectomized mouse model to mimic oxidative stress conditions in vivo.Serum malondialdehyde (MDA) and glutathione (GSH) levels were evaluated in ovariectomized mice with or without curcumin intervention.The results showed that the activity of MDA in serum increased in the ovariectomized group, whereas the GSH activity decreased compared to the control group.However, the serum activities of MDA and GSH were partially rescued by curcumin treatment (Figure 7A, 7B).

Curcumin improved bone mass and bone structure in ovariectomized mice
To further study the effects of curcumin treatment on trabecular bone mass and micro-architecture, different amounts of curcumin were delivered to the ovariectomized (OVX) mice intraperitoneally, and the trabecular bone mass and micro-architecture were evaluated with VG (Van Gieson) staining and microcomputed tomography (micro-CT) examination in all groups.As shown in (Figure 8A), control mice showed normal compact trabeculae in the femoral condyle, but sparse, thinned trabeculae were shown in OVX mice by VG staining.The disappearance and sparse trabeculae resulted in large spaces in the condyle.Curcumin treatment (5 μmol/kg, 15 μmol/kg) increased the thickness, density and the quantity of trabeculae.The bone mineral density and bone mass were evaluated by micro-CT examination (Figure 8B).Bone mass and bone trabeculae deteriorated in the ovariectomized mice, as measured through decreases in BMD, Tb.N, Tb.Th and BV/TV (Figure 8C-8F), but showed increases in BS/ TV,Tb.Sp and Tb.Pf (Figure 8G-8I).Curcumin treatment increased the bone micro-architecture and bone mineral density in a dose-dependent manner.These results confirmed that curcumin is able to reverse the in vivo bone formation reduction induced by oxidative stress.

DISCUSSION
Many different types of materials, such as metals, ceramics, and polymers, have been used in total hip replacements and spinal interbody fusion.Implanted materials can induce significant oxidative stress and inflammation around materials during their degradation, and material properties, such as their composition and their surface properties, also induced oxidative stress [21,22].ROS are considered the main type of cellular oxidant; many studies have indicated that increased oxidative stress is involved in the pathogenesis of bone metabolism [23], and the balance between oxidants and antioxidants may determine the fate of the implant materials.Reducing the production of ROS by using antioxidant drugs would be beneficial for osteogenesis.We chose curcumin, which has excellent antioxidant properties, as a candidate for  These results are consistent with previous studies [27,28].
Our previous study indicated that oxidative stress regulated the proliferation and osteogenic differentiation of MSCs through the miR-424/FGF2 pathway [26].Many studies have reported that oxidative stress inhibited differentiation of osteoblast-related cells, leading to reduce bone formation [29,30].Exogenous addition of H 2 O 2 to human BM-MSCs reduced ALP activity and abolished osteogenesis in osteoblast progenitors [31,32].Our experiments found that H 2 O 2 exposure reduced cellular ALP activity, calcium mineralization and expression of osteogenesis-related genes, including Alp, Col1a1,  Runx2 and Opn.Higher concentrations of curcumin also impaired ALP activity and mineralization, which was consistent with the above results.Moreover, our study showed that curcumin could reverse the H 2 O 2 -induced inhibition of osteogenic differentiation, and curcumin alone could enhance ALP activity and calcium levels in MC3T3-E1 cells at low concentrations.We found that 10 µM curcumin had the most obvious effect on improving osteoblastic differentiation in the face of oxidative damage, although the concentration was different from other studies, which may be due to the different cells involved and different experimental conditions [33].
Osteoblasts and osteoclasts are responsible for bone remodeling.RANKL is critical for the maturation and activity of osteoclasts, and IL-6 impaired bone formation via the dysfunction of osteoblasts.IL-6 could affect the expression of RANKL, leading to osteoclast development.
Several studies have reported that ROS could increase the expression of RANKL and IL-6 in osteoblasts [34,35].We observed that curcumin could reverse the effects of H 2 O 2 on the production of RANKL and IL-6, which may facilitate osteogenesis.
Our study showed that curcumin treatment reduced the H 2 O 2 -induced production of reactive oxygen species and partially reversed the inhibition of osteogenesis.However, the molecular mechanism of the curcumin effects in the MC3T3-E1 cells has not been established.To clarify the involved mechanism, we pre-treated the cells with curcumin for 24 h and then co-cultured them with H 2 O 2 for 1 h.Our study demonstrated that the production of ROS was increased to a greater extent with 1 h co-culture.Phospho-p65, p-P38, and p-JNK were constitutively active in H 2 O 2 -stimulated MC3T3-E1 cells; curcumin reduced the p-P65 expression and inhibited the phosphorylation of IκB-α, indicating that cells exposed to H 2 O 2 activated the NF-κB signaling pathway, which could be partially inhibited by curcumin.The concentration of 1 µM curcumin exerted the greatest inhibitory effect in our experiments.Consistent with our results, curcumin has been shown to inhibit the activation of NF-κB [36,37].Bharat et al. reported that curcumin reduced the activation of NF-κB and IκBα kinase in human multiple myeloma cells [38].In addition, curcumin alleviated oxidative stress in MC3T3-E1 cells by inhibiting P38 and JNK phosphorylation, and 20µM, 1 µM curcumin treatment demonstrated the greatest inhibitory effect respectively, the p-JNK, p-P65 other than p-P38 are the main signaling involved in accordance with the results of osteogenesis.In agreement with our study, curcumin inhibited oxLDLtriggered foam-cell formation through the p38 and JNK pathways [39][40][41].It has been reported that curcumin prevented diabetic cardiomyopathy by inhibiting the MAPK pathway in diabetic rats [42].Taken together, these findings indicated that curcumin might decrease oxidative damage by inhibiting NF-κB and MAPK signaling activity.
Many studies have shown that oxidative damage is the main culprit to bone loss in ovariectomized mice, which is considered the classic animal model mimicking the bone metabolism of postmenopausal osteoporosis [11,43,44].We therefore chose this animal model to in vivo investigate the pharmacological impact of curcumin on oxidative damage.Several studies have found that the production of MDA and GSH increases in the serum of postmenopausal women [45,46].Malondialdehyde (MDA) is a product of lipid peroxidation by reactive oxygen species; in contrast, glutathione (GSH), which removes free radicals, peroxides and toxins, is regarded as an intracellular antioxidant [47,48].We found that the activity of MDA increased and the activity of GSH decreased in the oxidative damage animal model, and curcumin treatment could effectively reverse these effects.
Similarly, the oxidative damage animal model also suffers significant bone loss, as shown by the micro-architecture of the trabecular bone; this has been termed "estrogen-deficient osteogenesis" [49][50][51].The micro-architecture of the trabecular bone was found to dramatically deteriorate with oxidative damage by both micro-CT scanning and VG staining.However, this process could be effectively reversed with curcumin treatment, and this protective effect was dose-dependent.Another study showed that curcumin does not have acute toxicity at daily doses of 2 g/kg of body weight in mice and 8,000 mg/day in humans for 3 months [52].These results further suggest that curcumin could ameliorate trabecular micro-architecture and bone mass deficiencies resulting from oxidative damage.
One of the limitations of the study is that although we investigated the mechanism involved in the protection of curcumin on the oxidative stress, we did not confirm the mechanism with signaling pathway inhibitor accordingly.The variation tendency of the key regulated factor of the pathway was target we focused on.Another limitation is that the ovariectomized mice were recruited to investigate the in vivo effect of the curcumin against the oxidative stress, and no materials were implanted in the animal model, because we thought the animal model could actually mimic the materials implanted environment.This study is our preliminary research, we intend to further verify our findings using an animal model with implantation of bone repair materials in the future study.
In conclusion, our studies indicate that oxidative stress due to H 2 O 2 treatment in vitro resulted in cytotoxicity and inhibited the osteogenic differentiation of MC3T3-E1 cells.Low concentrations of curcumin (1-10 µM) could reverse the oxidative damage, but a high concentration (20 µM) failed.Furthermore, the NF-κB and MAPK signaling pathways might be part of the potential mechanism involved in this protective process.In addition, curcumin reversed the ratio of oxidants and antioxidants, it improved the micro-architecture of the trabecular bone in an animal model of oxidative damage, and the effect is in a dose-dependent manner (Figure 9).Curcumin might therefore be a substantial alternative for prevention oxidative stress during material implanted, facilitating the bony fusion of artificial implanted devices.

Cell culture and treatment
Murine MC3T3-E1 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).Cells were cultivated in modified α-MEM medium with 10% heat-inactivated FBS (Gibco Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified 5% CO 2 atmosphere.The medium was replenished every three days.H 2 O 2 served as an exogenous ROS treatment, whereas N-acetyl-L-cysteine (NAC) served as an antioxidant.Fresh osteogenic medium (OM, 0.1 μM dexamethasone, 50 μg/ml ascorbic acid, and 10 mM β-glycerophosphate), containing different concentrations of curcuminwas used for osteogenic induction after 0.3 mM H 2 O 2 was administered for 24 h.All of the experiments were performed in duplicate wells and repeated three times.for 24 h in whole culture.Different concentrations of curcumin (1,5,10,20,50, 100 and 200 µM dissolved in 0.1% Dimethyl sulfoxide (DMSO) in distilled water) were also added to the whole cultures.A control group was treated with 0.1% DMSO in distilled water.The cells were incubated using whole culture medium, which contained curcumin (1,5,10,20,50, 100 or 200 µM) for 24 h followed by the administration of 300 µM H 2 O 2 for 24 h.The cells were rinsed with PBS once, and then, 120 μl of fresh medium containing 20 μl MTS was added to all wells.After incubation at 37°C for 3 h, the absorbance at 490 nm was measured using a spectrophotometric plate reader.

Alkaline phosphatase (ALP) staining and activity assay
The murine MC3T3-E1 cells were incubated in 96-well or 6-well plates overnight and treated with different concentrations of H 2 O 2 for 24 h, and/or the different concentrations of curcumin in osteogenic media throughout the differentiation process.After osteogenic induction for 7 days, the cells were lysed with 1% Triton X-100 overnight at 4°C.A portion of the cell lysate was incubated with p-NPP in a buffer (0.1 M glycine, 1 mMMgCl 2 , and ZnCl 2 , pH 10.3) at 37°C for 30 min; the reaction was terminated by adding 2 M NaOH, and the absorbance was measured at 405 nm.The ALP activity was normalized to total protein, which was measured using the Bradford protein assay.The cells were stained using the BCIP/NBT alkaline phosphatase color development kit according to the manufacturer's instructions.The assay was performed 7 days after osteogenesis.

Alizarin red staining and calcium accumulation assay
The mineralization of MC3T3-E1 cells was measured using Alizarin Red staining.The cells were incubated with osteogenic medium containing different concentrations of curcumin for 21 days after exposure to H 2 O 2 for 24 h.For Alizarin Red staining, cells were fixed with 4% paraformaldehyde for 15 min.The cells were stained with 40 mM Alizarin Red S for 15 min at room temperature.After rinsing with distilled water to completely remove the unbound stain, the cells were visualized and imaged using a light microscope (Nikon, Eclipse TS100, Japan) and digital camera (Nikon, D330, Japan).The calcium content was measured using a Calcium Assay kit at 21days according to the manufacturer's instructions.Briefly, samples were supplemented with 1 M acetic acid and placed on a vortex overnight at 4°C to extract the calcium from the mineralized matrix.In a 96-well clear polycarbonate plate, a portion of this cell extract was mixed with 150 μL Calcium Assay reagent and incubated for 30 s at room temperature.The absorbance at 575 nm was determined using a SpectraMAX 250 microplate reader.The samples were measured in triplicate and compared to a calcium calibration curve.The calcium content was normalized to cell protein levels and expressed as relative calcium content normalized to a control sample.

RNA isolation, cDNA synthesis and qRT-PCR.
Total RNA was extracted with TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer's instructions.Total RNA (500 ng of mRNA) from each sample was subjected to reverse transcription using a commercially available kit according to the manufacturer's protocol (TaKaRa, Japan).The primers used to amplify the osteogenic genes are listed in Table 1.All experiments were performed using a real-time PCR system (CFX ConnectTM Real-time system, Bio-Rad, USA).The expression levels of each mRNA were normalized to that of GAPDH.The relative expression levels of all genes were calculated using the 2 −ΔΔCt method.

RANKL and IL-6 measurements
After exposure to H 2 O 2 for 24 h, the murine MC3T3-E1 cells were incubated using whole culture medium, which contained different concentrations (1, 10, and 20 µM) of curcumin for 7 days.The production of RANKL and IL-6 in culture medium was tested using a sandwich ELISA assay kit according to the manufacturer's instructions.The total protein concentrations were measured using the Bradford protein assay method.

Intracellular ROS level determination
Intracellular ROS levels were evaluated using a method previously described with slight modification [53].Cells were seeded in 6-well plates at 2 × 10 5 cells/well.After culturing for 24 h in the incubator, different doses of H 2 O 2 were added.In brief, after the cells were exposed to H 2 O 2 for 1 h or 24 h, the cells were incubated in growth medium containing 20 μM DCFH-DA for 30 min at 37°C.The cells were then rinsed with PBS to remove the residual extracellular DCFH-DA.The cellular fluorescence was visualized using fluorescence microscopy (Axio Observer Z1, Carl ZeissInc.),or the cells were detached with trypsin and re-suspended after centrifugation.The fluorescence levels of the samples were measured using flow cytometry with the excitation and emission wavelengths set at 488 and 525 nm, respectively.

Preparation of cell lysates and western blot
Cells were harvested, and protein was extracted using RIPA buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich).The total protein concentration was determined with a BCA protein assay kit according to the manufacturer's instructions (Thermo Fisher Scientific, Rockford, IL, USA).Thirty micrograms of protein was separated by SDS-PAGE and then transferred onto a 0.45 μm PVDF membrane (Immobilon TM, Millipore Corp., Bedford, MA).The membranes were blocked and then probed overnight with rabbit polyclonal antibodies against p-p65, p-IκBα, p-JNK, JNK, p-P38, P38, p-ERK1/2, ERK1/2and β-actin.The antibodies were detected using enhanced chemiluminescence with HRP-conjugated secondary antibodies (Jackson Immuno Research).The values of the band intensities were quantified using Image J software.

Animals and curcumin intervention
The usage of animals were approved by the Institutional Animal Care and Use Committee at the University of Sun Yat-sen(permission code: SYXK2010-0108).Forty 8weeksold, BALB/c female mice, weighing about 20.52 ± 1.27 g, were obtained from the experimental animal center of the first affiliated hospital of Sun Yat-sen university (Guangzhou, China).There were no significant differences in the initial body weights of the mice among all 4 groups in the experiment.The mice were allowed to adapt to the laboratory environment (a well-ventilated controlled room maintained at 20°C on a 12 h light/dark cycle; the animals were given free access to water and food) for 7 days before the surgery.Subsequently, the mice underwent sham operation (n = 10) or ovariectomy (OVX) (n = 30) under anesthesia using sodium pentobarbital (50 mg/kg body weight, i.p.).The ovariectomy operation was performed with a dorsal approach.A total of 30 BALB/c female mice were randomly divided into three groups: 1) an OVX group, with DMSO solvent administered intraperitoneally (n = 10); 2) an OVX group, with curcumin solution administered intraperitoneally (5μmol/ kg body weight; n = 10) daily; and 3) an OVX group, with curcumin solution administered intraperitoneally (15μmol/ kg body weight; n = 10) daily.Curcumin was dissolved in distilled water with DMSO.One week after the operation, the treatments were initiated and continued for 12 weeks.Blood samples were obtained from the hearts of the anesthetized mice, and serum samples were prepared by centrifugation.The femurs of the mice were collected for further analysis, and the adherent tissue was discarded.

Measurements of serum malondialdehyde (MDA) andglutathione (GSH)
The MDA activity of the whole blood samples was determined using an MDA assay kit according to the manufacturer's instructions.Thiobarbituric acid binding to malondialdehyde, formed during lipid peroxidation, results in a chromogenic complex.In a spectrophotometer, the change in absorption peak was detected at 532 nm.Colorimetry was used to detect the malondialdehyde activity level in whole blood samples.Additionally, the activity of GSH was determined using a GSH assay kit.The GSH activity was determined by the reaction of GSH with dithio-bis-nitrobenzoic acid(DTNB) to produce a product that could be measured using a spectrophotometer at 412 nm.

Assessment of bone micro-architecture and bone mass by micro-computed tomography
The distal femurs were scanned using Inveon micro-CT/PET (Siemens Medical Solutions, Germany) with 15 µm resolution, 80 kV tube voltage and 500 μA tube current.The reconstruction and 3D quantitative analyses were determined using Inveon research workplace 4.1 software.Similar settings for scans and analyses were used for all of the samples.In the femur, the scanning regions were confined to the distal metaphysis, extending proximally 5.0 mm from the distal tip of the femoral condyle.The following 3D indices in the defined region of interest (ROI) were analyzed: bone mineral density (BMD), bone surface/total volume (BS/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular bone pattern factor (Tb.Pf) and relative bone volume/total volume (BV/TV,%).The operator who conducted the scan analysis was blinded to the procedure associated with the specimens.

Histological examination by Van Gieson (VG) staining
The femurs from all mice was collected and fixed in 4% paraformaldehyde for 48 h.After dehydration and embedding, the distal femurs were embedded in paraffin.The sections were cut and stained with VG staining, which was used to stain for collagen fibers according to the manufacturer's instructions, and examined by light microscopy.

Statistical analysis
The data are presented as the means ± SD.The differences between two groups were probed using the Student's t-test.The differences among multiple groups were evaluated using a one-way ANOVA.Statistically significant results were indicated as * P < 0.05, ** P < 0.01, # P < 0.05, and ## P < 0.01.Each experiment was repeated independently at least three times.

Figure 1 :
Figure 1: Levels of intracellular ROS in MC3T3-E1 cells and the cell viability under different concentrations of H 2 O 2 and curcumin.(A, B) The levels of intracellular ROS at 1 h and 24 h were determined using flow cytometry after MC3T3-E1 cells were incubated with H 2 O 2 at the above concentrations.(C) Effects of different concentrations of H 2 O 2 (0.1, 0.2, 0.3, 0.5, 1 and 2 mM) on the viability of cells cultured for 24 h or 48 h.(D) The viability of cells cultured with different concentrations of curcumin (0,1, 5, 10, 20, 50, 100 and 200 μM) for 24 h and 48 h.(E) The viability of cells exposed to 0.3 mM H 2 O 2 for 24 h after pretreatment with different concentrations of curcumin for 24 h.Data are presented as the means ± S.E.M. * p < 0.05 and ** p < 0.01 versus control.

Figure 3 :
Figure 3: Protection by curcumin on the mRNA expression of alp, col1a1, runx2 and opn after exposure to H 2 O 2 for 24 h.MC3T3-E1 cells were induced for 7 days with different concentrations of curcumin.(A, B) Relative Alp, Collagen I mRNA expression (C, D) Relative Runx2 and Opn mRNA expression.Gene expression was normalized to GAPDH.# P < 0.05 and ## P < 0.01 compared to control group; * P < 0.05 and ** P < 0.01 compared to the H 2 O 2 group.

Figure 4 :
Figure 4: Curcumin down-regulated the expression of IL-6 and RANKL.After exposure to H 2 O 2 for 24 h, MC3T3-E1 cells were induced for 7 days with different concentrations of curcumin.(A) The relative expression of RANKL in the presence of curcumin and/ or H 2 O 2 .(B) The relative expression of IL-6 in the presence of curcumin and/or H 2 O 2 .## P < 0.01 compared to the control group; * P < 0.05 and ** P < 0.01 compared to the H 2 O 2 alone treatment group.

Figure 7 :
Figure 7: Curcumin attenuates oxidative stress in ovariectomized mice.(A) Serum MDA concentrations in sham operation and OVX-operation groups treated with or without curcumin.(B) Serum GSH concentrations in sham and OVX-operation groups treated with or without curcumin.##P < 0.01 compared to the sham group; * P < 0.05 and ** P < 0.01 compared to the OVX group.

Figure 9 :
Figure 9: A schematic representation of the main conclusions of this study.