Curcumin Ameliorates Palmitic Acid-Induced Saos-2 Cell Apoptosis Via Inhibiting Oxidative Stress and Autophagy

Objectives We aimed to determine the effects of curcumin on palmitic acid- (PA-) induced human osteoblast-like Saos-2 cell apoptosis and to explore the potential molecular mechanisms in vitro level. Methods Saos-2 cell were cultured with PA with or without curcumin, N-acetylcysteine (NAC, anti-oxidant), 3-methyladenine (3-MA, autophagy inhibitor) AY-22989 (autophagy agonist) or H2O2. Then, the effects of PA alone or combined with curcumin on viability, apoptosis, oxidative stress, and autophagy in were detected by CCK-8, flow cytometry assay and western blot. Results We found that autophagy was induced, oxidative stress was activated, and apoptosis was promoted in PA-induced Saos-2 cells. Curcumin inhibited PA-induced oxidative stress, autophagy, and apoptosis in Saos-2 cells. NAC successfully attenuated oxidative stress and apoptosis, and 3-MA attenuated oxidative stress and apoptosis in palmitate-induced Saos-2 cells. Interestingly, NAC inhibited PA-induced autophagy, but 3-MA had no obvious effects on oxidative stress in PA-treated Saos-2 cells. In addition, curcumin inhibited H2O2 (oxidative stress agonist)-induced oxidative stress, autophagy, and apoptosis, but curcumin had no obvious effect on AY-22989 (autophagy agonist)-induced autophagy and apoptosis. Conclusion The present study demonstrated that oxidative stress is an inducer of autophagy and that curcumin can attenuate excess autophagy and cell apoptosis by inhibiting oxidative stress in PA-induced Saos-2 cells.


Introduction
Diabetes mellitus is a pandemic metabolic disease and has a worldwide distribution. Patients with diabetes mellitus have various skeletal disorders, including osteopenia or osteoporosis [1]. Diets rich in high-fat foods, especially saturated fats, are usually the cause of the clinical symptoms of metabolic syndrome, such as obesity, insulin resistance, and type 2 diabetes, which eventually increase the likelihood of osteoporosis [2]. Moreover, obesity and type 2 diabetes trigger a prolonged elevation of circulating free fatty acid levels (FFAs) especially the saturated FFAs such as palmitate (PA), which causes lipotoxicity in many cell types, including human osteoblast-like Saos-2 cell [3]. Additionally, PA-induced lipotoxicity plays a vital role in the development and progression of osteoporosis [4,5]. Numerous studies have focused on factors involved in the mechanism of PA-induced lipotoxicity, such as oxidative stress and autophagy [5].
Oxidative stress is essentially an imbalance between the generation of reactive oxygen species (ROS) and the ability of the body to counteract or detoxify their harmful effects through neutralization by antioxidants [6]. ROS are produced in all cellular compartments as a result of exposure to toxic agents and natural by-products of mitochondrial respiration and can disrupt the normal mechanisms of cellular signaling and function, resulting in DNA damage and apoptosis [7]. Previous studies have reported that oxidative stress plays an important role in the pathophysiology of many diseases, including osteoporosis [6].
Autophagy is a complex catabolic process in eukaryotes that enables cells to recycle cytoplasmic components through degradation in the lysosomes. Under stressful conditions, such as nutrient deprivation and oxidative stress, autophagy is activated as a pathway to promote cell survival by maintaining energy and reducing toxic substances [8]. In addition, there is increasing evidence that excessive or uncontrolled levels of autophagy may be essential for cell apoptosis in certain settings [9]. Moreover, some studies have reported that autophagy is related to diabetic osteoporosis, [8] and oxidative stress has been reported to be a novel autophagy inducer [10].
Curcumin, a non-flavonoid polyphenol found in the plant Curcuma longa, has been extensively investigated because of its anti-inflammatory, anti-oxidative, and cytoprotective properties [11]. Previous studies have reported that curcumin is a promising drug for the prevention and treatment of diabetes and diabetes-related diseases, including osteoporosis [12]. Moreover, both oxidative stress and autophagy are related to diabetic osteoporosis [13]. In addition, previous study has reported that curcumin can regulate oxidative stress and autophagy in vivo and in vitro [14].
In this study, we aimed to determine the effects of curcumin on PA-induced human osteoblast-like Saos-2 cell apoptosis and to explore the potential molecular mechanisms in vitro level. Herein, we investigated the participation and relationship of oxidative stress and autophagy and evaluated the effects and molecular mechanisms of curcumin in PA-induced Saos-2 cell apoptosis.

Cell Culture and Treatment.
Saos-2 cells were cultured in DMEM supplemented with 10% FBS, 50 μg/mL penicillin, and 50 μg/mL gentamicin in a humidified incubator at 37°C with 5% CO 2 . When the cells reached 70%-80% confluence, they were treated with different concentrations (1.25-20 μM) of curcumin and 200 µM PA to determine their effects on cell activity. At the same time, the cells were exposed to 200 µM PA in the presence or absence of 10 µM curcumin, 5 mM 3-MA, 10 µM AY-22989, 100 µM H 2 O 2 , or 2 mM NAC for 24 h.

Measurement of Cell Viability.
After the treatment, 10 μl of CCK-8 was added to each well, and the cells were incubated for 2 h at 37°C. e number of viable cells was measured at 450 nm with a microplate reader (Bio-Rad 680). e results are presented as a percentage of the values measured for untreated control cells.

Cell Apoptosis Measurement.
After the treatment, cell apoptosis was quantified with an Annexin V-FITC/PI Apoptosis Analysis Kit according to the manufacturer's instructions. e cells were analyzed using a flow cytometer (Becton, Dickinson and Company, USA) within 1 h. Early apoptotic cells were determined by counting the percentage of Annexin V-FITC+/PI− cells, progressed apoptotic cells were obtained by counting the percentage of Annexin V-FITC+/PI+ cells, and necrotic cells were detected by counting the percentage of Annexin V-FITC−/PI+ cells and Annexin V-FITC−/PI− cells.

Western Blot.
After the treatment, cells were collected and washed with ice-cold PBS and lysed with RIPA buffer, and the total protein concentration was measured using the BCA assay. Fifty micrograms of total protein from each sample were loaded into each well of a 13% SDS-PAGE gel, and then, proteins were separated by electrophoresis. Proteins were then transferred to PVDF membranes. After blocking in TBST with 10% nonfat milk for 2 h, the samples were incubated with primary antibody against B-cell lymphoma-2 (Bcl-2, 1 : 1,000), Bcl-2 associated X protein (BAX, 1 : 500), glyceraldehyde-phosphate dehydrogenase (GAPDH, 1 : 2,000), Autophagy protein 5 (ATG5, 1 : 1,000), polyubiquitin-binding protein (P62, 1 : 1,000), and microtubule-associated protein light chain 3 (LC3, 1 : 1,000) overnight at 4°C. After washing, the membranes were incubated with secondary antibody (1 : 4,000) conjugated to horseradish peroxidase at 37°C for 30 min. e immunoreactive bands were visualized using a Super Signal West Pico kit according to the manufacturer's instructions, and the densities of the protein bands were measured by densitometric analysis using ImageJ software 1.48 (National Institutes of Health, Bethesda, Maryland, USA).

mCherry-GFP-LC3
Adenovirus Transfection and Autophagy Assay. Cells cultured in 24-well plates (1 × 105 cells/ well) were transfected with mCherry-GFP-LC3 adenovirus at 40 MOI for 24 h. After transfection, the cells were incubated with fresh culture medium again for 24 h. e numbers of GFP and mCherry dots per cell were counted in three randomly selected fields under a fluorescence microscope (Olympus Corporation, Tokyo, Japan). GFP degrades in an acidic environment, while mCherry does not. us, yellow spots (formed from the overlap of red and green) indicate autophagosomes, while red spots indicate autophagic lysosomes. If autophagy is activated, the red signal will dominate over yellow. If autophagy is suppressed, there will be more yellow signal than red signal.

ROS Measurements.
After the treatment, the cells were incubated with DCFH-DA (10 µM) at 37°C for 20 min. en, the cells were subsequently washed three times with PBS, and the ROS-sensitive dye in cells was assessed under an inverted fluorescence microscope (Olympus Corporation, Tokyo, Japan) with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Fluorescent intensity was acquired using ImageJ 1.48 image analysis software.

Superoxide Dismutase (SOD) Detection.
After the treatment, the cells were harvested and sonicated in PBS containing 1.0 mM PMSF to obtain cell homogenates. e homogenates were then centrifuged at 15,000 × g and 4°C for 15 min, and the protein content was determined using the BCA Protein Assay Kit. en, the supernatants were used for measuring cellular SOD. e SOD activity was determined at 450 nm using a microplate reader (Bio-Rad 680).

Caspase-3 Activity Measurement.
After the treatment, the cells were harvested by centrifugation and incubated in lysis buffer on ice for 15 min. e lysates were then centrifuged at 15,000 × g and 4°C for 15 min, and the protein content was determined using the BCA Protein Assay Kit according to the manufacturer's instructions. en, each sample was incubated with the Caspase-3 substrate at 37°C in a microplate for 4 h. e samples were measured at 405 nm using a microplate reader.

Statistical Analyses.
All experiments were repeated at least three times for each group, and the data are expressed as the mean ± standard error of the mean (SEM). e data were analyzed by one-way analysis of variance (ANOVA) followed by Fisher's least significant difference test and independent samples Student's t-test with SPSS software, version 13.0 (SPSS, Chicago, IL, USA). Differences were considered statistically significant when P < 0.05. Flow cytometry analysis revealed that curcumin prevented cell apoptosis caused by PA in a dose-dependent manner. However, 20 μM curcumin had no obvious effects on PAinduced cell apoptosis (Figure 1(c)). e colorimetric assay and western blot results showed that curcumin attenuated the expression of BAX and Caspase-3 but increased the expression of Bcl-2 in PA-treated Saos-2 cells (Figures 1(d) and 1(e)).

Curcumin Reduced PA-Induced Oxidative Stress and Autophagy in Saos-2 Cells.
To determine the effects of curcumin on oxidative stress and autophagy in PA-treated Saos-2 cells, the cells were treated with 200 µM PA with or without 1.25-10 μM curcumin for 24 h, and oxidative stressrelated parameters were analyzed. DCFH-DA assay revealed that PA increased intracellular ROS production, but curcumin treatment decreased the PA-induced ROS generation in a dose-depend manner (Figure 2(a)). Moreover, PA reduced the activity of the anti-oxidant enzyme SOD, which was rescued by 1.25-10 μM curcumin in a dose-dependent manner ( Figure 2(b)). ese results showed that curcumin attenuates PA-induced cell apoptosis in Saos-2 cells. e western blot results indicated that the 200 μM PA treatment increased the protein expression of ATG5 and LC3II and the degradation of p62. Meanwhile, we found that the protein expression levels of ATG5 and LC3II in PA-treated cells significantly decreased but that of p62 increased following the addition of different doses of curcumin (Figures 2(c)-2(f )).

Roles of Oxidative Stress and Autophagy in the Protective Effect of Curcumin on PA-Induced Saos-2 Cell Apoptosis.
Because both oxidative stress and autophagy can be induced during Saos-2 cell apoptosis, we designed the following experiment to determine whether oxidative stress and autophagy are involved in PA-induced Saos-2 cell apoptosis. e oxidative stress inhibitor NAC and autophagy inhibitor 3-MA were added to the culture medium during the PA treatment. e CCK-8 assay results showed that NAC and 3-MA clearly increased cell viability in PA-induced Saos-2 cells (Figure 3(a)). e flow cytometry results indicated that NAC and 3-MA reduced cell apoptosis caused by PA (Figures 3(b) and 3(c)). e western blot and colorimetric assay results demonstrated that the decreases in BAX and Caspase-3 expression and the increase in Bcl-2 expression were also rescued by NAC and 3-MA in PA-induced Saos-2 cells (Figures 3(d) and 3(e)). Moreover, NAC and 3-MA had the same protective effect as curcumin on PA-induced Saos-2 cell apoptosis. erefore, these results indicate that PA induces Saos-2 cell apoptosis by stimulating oxidative stress and activating autophagy and that curcumin may attenuate cell apoptosis by inhibiting oxidative stress and autophagy.

Relation of Oxidative Stress and Autophagy to the Protective Effect of Curcumin on PA-Induced Saos-2 Cell.
To determine whether there is an association between oxidative stress and autophagy in PA-induced Saos-2 cells, we assessed the oxidative stress parameters and autophagy makers in PA-induced Saos-2 cells with or without NAC and 3-MA. e oxidative stress parameter results indicated that NAC, but not 3-MA, inhibited the ROS generation and SOD downregulation (Figures 4(a)-4(c)), suggesting that NAC, but not 3-MA, abrogated PA-induced oxidative stress. In addition, NAC and 3-MA clearly reduced the protein levels of ATG5 and LC3II and the degradation of P62 (Figures 4(d)-4(g)). Moreover, the NAC and 3-MA treatments inhibited the PA-induced increases in autophagosomes and autolysosomes (Figures 4(h) and 4(i)), suggesting that NAC and 3-MA alleviated PA-activated autophagy. erefore, NAC, but not 3-MA, had the same effect as curcumin on PA-induced oxidative stress and autophagy in Saos-2 cells. ese results indicated that oxidative stress is an inducer of autophagy in PA-induced Saos-2 cells and that curcumin may reduce oxidative stress and oxidative stressinduced autophagy in PA-induced Saos-2 cells.

Discussion
In the past few decades, with the dramatic increase in the type 2 diabetes epidemic, our understanding of the role of dyslipidemia and lipotoxicity in many diseases has grown considerably. e association between hyperlipidemia and osteoporosis has been established clinically and in experimental models [15]. Palmitate is the main saturated FFA in plasma that stimulates ROS production and autophagy activity in cultured cardiomyocytes and endothelial cells [16]. In our previous study, we reported that PA triggers Saos-2 cell apoptosis via excessive autophagy [3], and several studies have reported that curcumin protects cells against oxidative stress-mediated apoptosis [14,17]. However, the cryoprotective effect of curcumin on PA-treated cells and the underlying mechanisms have not been reported. Here, we demonstrated for the first time that curcumin can attenuate PA-induced Saos-2 cell apoptosis via inhibiting oxidative stress and autophagy (Figure 7).

Evidence-Based Complementary and Alternative Medicine
Curcumin has a range of pharmacological effects, including anticancer, anti-inflammatory, antioxidative as well as antioxidant, and cryoprotective activities [18,19]. In our research, curcumin rescued the PA-induced cell viability decrease and cell apoptosis. Our results are different from those of previous studies that reported that curcumin triggers apoptosis in many tumor cells, including HT29 cells [20] and upper aerodigestive tract cancer cells [21], suggesting that curcumin is a potential anticancer drug. Meanwhile, our results further supported the conclusion that curcumin protects cells against various injuries such as ischemic injury [18,22] and diabetes [23,24] in vivo and in vitro. erefore, curcumin seems to have a bidirectional effect on cell proliferation and apoptosis. On the one hand, it can promote cell apoptosis, especially in cancer cells; on the other hand, it can prevent cell apoptosis. e reasons for the different results (cell die or live) caused by curcumin may be due to differences in cell types, drug doses, and experimental methods. 8

Evidence-Based Complementary and Alternative Medicine
We further examined apoptotic regulatory genes, including Caspase-3, BAX, and Bcl-2. e results showed that curcumin decreased the expression of the pro-apoptotic molecule BAX and increased the expression of the antiapoptotic molecule Bcl-2. Our results were consistent with the findings of previous studies that reported that curcumin ameliorated cell apoptosis by regulating Bcl-2 family proteins, which can downregulate BAX and upregulate Bcl-2 expression [25]. Caspase-3 acts as an executioner in Caspase-mediated apoptosis, and the expression of Caspase-3 positively correlates with the rate of apoptosis in cells [11]. In our study, we found that curcumin can inhibit Caspase-3 activity, suggesting that curcumin can maintain cell survival by restraining Caspase-3. erefore, we propose that curcumin affects cell proliferation and apoptosis in PA-treated cells by regulating Bcl-2 family proteins and mitigating Caspase-3 activity.
In previous studies, oxidative stress was found to be involved in the process of PA-mediated apoptosis [7,26]. In addition, ROS production is a particularly destructive aspect of oxidative stress. Apoptosis is induced by excess free radicals when ROS production exceeds the capacity of antioxidant defenses [7]. In our study, we found that PA enhanced ROS generation. SOD is an antioxidant that protect cells against oxidative damage. However, the downregulation of SOD further promotes oxidative stress and damages cells. Previous studies have demonstrated that curcumin can prevent cell damage via suppressing oxidative stress [27]. Our data indicated that curcumin alleviates oxidative stress in a dose-dependent manner, indicating the protective ability of curcumin to prevent PA-induced oxidative stress in Saos-2 cells.
Autophagy is an important intracellular bulk degradation process involving the lysosome-dependent turnover of damaged cytosolic proteins and organelles, and it is critical for the maintenance of the normal cell phenotype and its functions. However, excess autophagy triggers cell apoptosis by destroying large proportions of the cytosol and organelles [28]. In our previous study we found that PA activated autophagy in a dose-and time-dependent manner in Saos-2 cells. Moreover, the PA-induced excessive autophagy caused cell apoptosis in Saos-2 cells. Furthermore, we analyzed the effects of curcumin on PA-induced autophagy. e results showed that PA successfully induced the expression of autophagy-related genes and that curcumin inhibited autophagy in a dose-dependent manner, suggesting that curcumin blocked.
To clarify whether oxidative stress and autophagy triggered PA-induced Saos-2 cell apoptosis, we used the oxidative stress inhibitor NAC and the autophagy inhibitor 3-MA to treat Saos-2 cells during the PA treatment. Apoptosis was inhibited by NAC and 3-MA.
ese results further indicated that PA-induced oxidative stress and autophagy cause Saos-2 cell apoptosis. Together, the results that curcumin inhibits autophagy, oxidative stress, and apoptosis indicate that curcumin may attenuate cell apoptosis by inhibiting oxidative stress and autophagy in PA-induced Saos-2 cells.
Previous studies have shown that oxidative stress can induce autophagy under starvation conditions [29]. Consistent with this previous study, we found that H 2 O 2 activated autophagy in Saos-2 cells. Moreover, the PA-induced autophagy activity was inhibited by NAC in Saos-2 cells. In addition, AY-22989 and 3-MA did not have an effect on Saos-2 cells with or without PA treatment.
ese results indicated that oxidative stress is an inducer of autophagy in      PA-induced Saos-2 cells. However, it is still unclear whether curcumin directly affects autophagy in PA-induced Saos-2 cells. Moreover, some studies have also reported that curcumin induces autophagy activation, such as in osteosarcoma MG63 cells [30] and oral cancer cells [31].
To further determine the effects of curcumin on autophagy in Saos-2 cells, we used AY-22989 to activate autophagy during the treatment. e results showed that curcumin had no obvious effects on autophagy and autophagy-induced apoptosis, indicating that curcumin has no effect on autophagy in Saos-2 cells. In addition, we found that the apoptosis, oxidative stress, and autophagy induced by H 2 O 2 were rescued by curcumin, suggesting that curcumin can inhibit oxidative stress-induced autophagy and apoptosis in Saos-2 cells.
In summary, we found that curcumin can inhibit PAinduced oxidative stress, autophagy, and apoptosis. Moreover, previous studies have reported that oxidative stress can activate these processes in many cells and tissues [7,32]. Our results showed that NAC can inhibit PA-induced autophagy but that 3-MA had no obvious effects on oxidative stress in Saos-2 cells with PA treatment. us, we assume that curcumin indirectly inhibits the PA-induced autophagy via suppressing oxidative stress. To further clarify this hypothesis, we found that curcumin inhibited H 2 O 2 -induced oxidative stress, autophagy, and apoptosis, but curcumin had no obvious effects on AY-22989-induced autophagy. erefore, curcumin ameliorates PA-induced human Saos-2 cell apoptosis by inhibiting oxidative stress-mediated autophagy. However, there were still a notable limitation in our study. e effects of curcumin on the lipotoxicity of Saos-2 cell in our present study might not represent the actual condition of human diabetic osteoporosis. erefore, this study is the first step toward assessing the use of curcumin as a bioagent for the treatment of diabetic osteoporosis. To this end, we intend to establish an animal model to further confirm our results with the same way.

Conclusions
is is the first study to report that curcumin protects against PA-induced human Saos-2 cell apoptosis via inhibiting oxidative stress and autophagy (Figure 7). Our result may offer new insights into the molecular mechanisms and treatment of lipotoxicity in diabetic osteoporosis.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.