7-Ketocholesterol Induces Oxiapoptophagy and Inhibits Osteogenic Differentiation in MC3T3-E1 Cells

7-Ketocholesterol (7KC) is one of the oxysterols produced by the auto-oxidation of cholesterol during the dysregulation of cholesterol metabolism which has been implicated in the pathological development of osteoporosis (OP). Oxiapoptophagy involving oxidative stress, autophagy, and apoptosis can be induced by 7KC. However, whether 7KC produces negative effects on MC3T3-E1 cells by stimulating oxiapoptophagy is still unclear. In the current study, 7KC was found to significantly decrease the cell viability of MC3T3-E1 cells in a concentration-dependent manner. In addition, 7KC decreased ALP staining and mineralization and down-regulated the protein expression of OPN and RUNX2, inhibiting osteogenic differentiation. 7KC significantly stimulated oxidation and induced autophagy and apoptosis in the cultured MC3T3-E1 cells. Pretreatment with the anti-oxidant acetylcysteine (NAC) could effectively decrease NOX4 and MDA production, enhance SOD activity, ameliorate the expression of autophagy-related factors, decrease apoptotic protein expression, and increase ALP, OPN, and RUNX2 expression, compromising 7KC-induced oxiapoptophagy and osteogenic differentiation inhibition in MC3T3-E1 cells. In summary, 7KC may induce oxiapoptophagy and inhibit osteogenic differentiation in the pathological development of OP.


Introduction
Osteoporosis (OP), commonly marked by a constant decrease in bone mass and changes in bone/skeletal tissue structure, is a common bone/skeletal disease [1]. More often than not, patients do not realize OP at the early stage until accidental fractural damages occur [2]. The pathogenesis and progression of OP might be affected by aging, genetic factors, bad living habits, and nutritional deficiency, among which, the imbalance of bone homeostasis is one of the key factors [3,4]. There is increasing evidence that lipid metabolism disorders, such as atherosclerosis (AS) and OP, share common underlying pathogenesis involving bone and vascular mineralization, as well as age-related degenerative processes [5][6][7]. However, the specific mechanism of action between lipid and bone metabolism disorders remains unclear.
Oxysterols are the lipid oxidation products of 27C cholesterol obtained from certain diets and cholesterol metabolism by enzymatic or non-enzymatic mechanisms [8]. Endogenous oxysterols are generated by the latter mechanism, with oxidation occurring in the sterol ring. Many factors, such as singlet oxygen and hydrogen peroxide, can induce the oxidation of cholesterol in lipoproteins, cell membranes, and food [9]. Oxysterols can be formed in foods containing cholesterol during prolonged storage or heating. In addition, cholesterol in the stomach can also be transformed into oxidized cholesterol due to an acidic pH and the presence of oxygen and iron ions [10]. Oxysterols can trigger oxidation, and washed twice with PBS. The solution contained 10 µM of 2 ,7 -Dichlorodihydrofluorescein diacetate (DCFH-DA, Solarbio, Beijing, China) in a serum-free medium and was incubated for 30 min in the dark. The cells were washed in PBS three times. Next, 4% paraformaldehyde (Beyotime, Shanghai, China) was added to the system and the cells were fixed at room temperature for 10 min. After the cells had been washed in PBS three times, 4',6-diamidino-2-phenylindole (DAPI, Solarbio, Beijing, China) was used for staining for 10 min, the cells were washed three times with PBS again, and the fluorescence intensity was observed using a fluorescence microscope and photographed. ImageJ/Fiji, an open-source image processing package, was used to analyze the mean fluorescence intensity.

Apoptosis Analysis
The apoptosis rates were determined by employing the Annexin V apoptosis kit which was obtained from BD Biosciences (San Jose, CA, USA). The cells (5 × 10 5 cells/wells) were grown until confluence of about 80% was reached. After the proper treatment, the cells were collected. Under the guideline of the kit's instructions, the cells were moved for apoptotic determination by flow cytometry (BD FACS Canto II, San Jose, CA, USA).

ALP Staining
MC3T3-E1 cells were inoculated with 1 × 10 5 cells/wells and cultured for 24 h before experiments. After 14 days of incubation, cells were collected and washed with the Beyotime. The ALP activity was then measured using an ALP activity assay kit (Beyotime). Finally, the absorption at the 405 nm wavelength was measured under the guidelines of a microplate reader (Varioskanlux, Thermo Fisher Scientific, Waltham, MA, USA).

Determination of MDA and SOD Activities
MC3T3-E1 cells were inoculated with 2 × 10 6 cells/wells for 24 h before experiments. Then, the cells were harvested and lysed on ice in RIPA Lysis Buffer (Beyotime) for 30 min. The lysates were collected after centrifugation, and the protein concentration was determined. Then, the SOD and MDA activities were measured using a SOD kit (Beijing Solarbio, China) and an MDA kit (Beijing Solarbio, China). Finally, the absorption of SOD and MDA at 560 nm and 532 nm, respectively, was measured under the guidelines of a microplate meter (Varioskanlux).

Mineralization Detection
MC3T3-E1 cells (1 × 10 5 cells/well) were cultured for 24 h. After 14 days of incubation, the cells were washed and fixed with 4% paraformaldehyde (Beyotime, Shanghai, China). Then, the cells were stained with alizarin red S (Solarbio, Beijing, China) at 37 • C for 30 min. After washing with PBS, orange calcified nodules were observed under an inverted microscope. Data were analyzed under the guideline of the kit's instructions.

Statistical Analysis
Data were indicated as the mean ± standard deviation (SD). Statistical analysis was conducted by the GraphPad Prism v7 software (GraphPad Software Inc., La Jolla, CA, USA). The one-way analysis of variance (ANOVA) and subsequent Bonferroni's multiple comparisons test were analyzed. p < 0.05 indicated a statistical difference.
MC3T3-E1 cells (1 × 10 5 cells/well) were cultured for 24 h. After 14 days of incubation, the cells were washed and fixed with 4% paraformaldehyde (Beyotime, Shanghai, China). Then, the cells were stained with alizarin red S (Solarbio, Beijing, China) at 37 °C for 30 min. After washing with PBS, orange calcified nodules were observed under an inverted microscope. Data were analyzed under the guideline of the kit's instructions.

Statistical Analysis
Data were indicated as the mean ± standard deviation (SD). Statistical analysis was conducted by the GraphPad Prism v7 software (GraphPad Software Inc., La Jolla, CA, USA). The one-way analysis of variance (ANOVA) and subsequent Bonferroni's multiple comparisons test were analyzed. p < 0.05 indicated a statistical difference.

7KC Inhibited the Osteogenic Differentiation of MC3T3-E1 Cells
In order to explore the biological actions of 7KC on MC3T3-E1 cell differentiation, ALP activity detection and mineral assays were conducted. Results showed that 7KC significantly attenuated ALP activity and alizarin red S staining (

7KC Inhibited the Osteogenic Differentiation of MC3T3-E1 Cells
In order to explore the biological actions of 7KC on MC3T3-E1 cell differentiation, ALP activity detection and mineral assays were conducted. Results showed that 7KC significantly attenuated ALP activity and alizarin red S staining (

7KC Induced Oxiapoptophagy in MC3T3-E1 Cells
7KC is proven to produce oxiapoptophagy [19,21,31]. To explore the biological actions of 7KC on MC3T3-E1 cells, DCFH-DA staining was employed, and the results show that 7KC increased ROS levels in MC3T3-E1 cells in a concentration-dependent manner

7KC Induced Oxiapoptophagy in MC3T3-E1 Cells
7KC is proven to produce oxiapoptophagy [19,21,31]. To explore the biological actions of 7KC on MC3T3-E1 cells, DCFH-DA staining was employed, and the results show that 7KC increased ROS levels in MC3T3-E1 cells in a concentration-dependent manner ( Figure 3A,B). The SOD activity and MDA level of MC3T3-E1 cells treated by 7KC significantly decreased ( Figure 3C,D). NADPH oxidase 4 (NOX4) plays an important role in the production of ROS, and it has been reported that 7KC can increase NOX4 expression in vascular smooth muscle cells [32,33]. Here, 7KC stimulated NOX4 expression in MC3T3-E1 cells ( Figure 3E,F). In addition, 7KC increased the ratio of LC3-II/LC3-I protein expression, enhanced Beclin 1 expression, and significantly decreased P62 protein expression ( Figure 3G-J). 7KC treatment also resulted in an increased apoptosis-related protein Bax/Bcl-2 ratio and cleaved caspase-3 protein expression ( Figure 3K-M). Flow cytometry showed that 7KC could significantly increase the apoptosis of MC3T3-E1 cells ( Figure 3N,O). Collectively, treatment with 7KC in MC3T3-E1 cells might induce oxidative stress, autophagy, and apoptosis, known as oxiapoptophagy.

Discussion
Previous studies indicate a correlation of AS with an enhanced fracture risk for bone [5]. However, the biological action of oxysterol, a critical regulator implicated in the pathophysiological process of AS, on bone metabolism has not been fully elucidated. Current results indicate that 7KC lowered the viability, increased the generation of ROS, induced oxiapoptophagy, and attenuated the osteogenic differentiation in pre-osteoblast MC3T3-E1 cells. In addition, pre-incubation with NAC strongly attenuates 7KC-induced pathological changes.
Oxysterols are considered to be a class of factors associated with human physiological and pathological processes. For example, oxysterols were recently demonstrated to function as the ligands that interact with the liver X receptors (LXRα/β), which are involved in the regulation of cholesterol metabolism by mediating the transcription of specific genes, such as ABCA1, ABCG1, and SREBP-1c [34]. Oxysterols may exhibit pro-apoptotic and pro-autophagic activities, depending on the difference in oxysterol types, cell lines, and treatment concentrations [35]. The main types of cell death induced by oxysterols, apoptosis, autophagy, and necrosis, have been studied [10,36]. However, cell necrosis may only be induced by high concentrations of oxysterols in some cell lines [37]. It has been demonstrated that 27-hydroxycholesterol may decrease osteoblast differentiation, increase osteoclastogenesis, and increase bone resorption by interacting with estrogen receptors and LXRs [38]. 25-Hydroxycholesterol is reported to increase apoptosis, promote osteogenic differentiation, and induce vascular calcification in vascular smooth muscle cells (VSMCs) by triggering endoplasmic reticulum stress [39]. 7KC is reported to promote cell apoptosis in mesenchymal stem cells derived from adipose tissue [40]. Similarly, 7KC

Discussion
Previous studies indicate a correlation of AS with an enhanced fracture risk for bone [5]. However, the biological action of oxysterol, a critical regulator implicated in the pathophysiological process of AS, on bone metabolism has not been fully elucidated. Current results indicate that 7KC lowered the viability, increased the generation of ROS, induced oxiapoptophagy, and attenuated the osteogenic differentiation in pre-osteoblast MC3T3-E1 cells. In addition, pre-incubation with NAC strongly attenuates 7KC-induced pathological changes.
Oxysterols are considered to be a class of factors associated with human physiological and pathological processes. For example, oxysterols were recently demonstrated to function as the ligands that interact with the liver X receptors (LXRα/β), which are involved in the regulation of cholesterol metabolism by mediating the transcription of specific genes, such as ABCA1, ABCG1, and SREBP-1c [34]. Oxysterols may exhibit pro-apoptotic and pro-autophagic activities, depending on the difference in oxysterol types, cell lines, and treatment concentrations [35]. The main types of cell death induced by oxysterols, apoptosis, autophagy, and necrosis, have been studied [10,36]. However, cell necrosis may only be induced by high concentrations of oxysterols in some cell lines [37]. It has been demonstrated that 27-hydroxycholesterol may decrease osteoblast differentiation, increase osteoclastogenesis, and increase bone resorption by interacting with estrogen receptors and LXRs [38]. 25-Hydroxycholesterol is reported to increase apoptosis, promote osteogenic differentiation, and induce vascular calcification in vascular smooth muscle cells (VSMCs) by triggering endoplasmic reticulum stress [39]. 7KC is reported to promote cell apoptosis in mesenchymal stem cells derived from adipose tissue [40]. Similarly, 7KC can induce oxiapoptophagy in BMSCs from patients with acute myeloid leukemia by mediating the Sonic Hedgehog pathway [19]. However, whether 7KC affects osteogenic differentiation by inducing oxiapoptophagy is still unclear.
To investigate the toxicity of 7KC on MC3T3-E1 cells, we measured the effect of 7KC on cell viability. We found that 7KC exhibited cytotoxicity at a concentration as low as respectively. It is reported that 7KC can induce cytotoxicity at a concentration range of 5 to 80 µg/mL in different cell lines in 48 h [41]. In U937 cells, 7KC induces cell apoptosis with maximal proportions (40 ± 5%) at the concentration of 40 µg/mL in 30 h [42]. In VSMCs, 7KC promotes cell apoptosis at a high concentration of 30 µM in 6 days [43]. In human umbilical venous endothelial cells, 7KC at concentrations of 10 µg/mL and 20 µg/mL does not produce effects on cell apoptosis. At a concentration of 40 µg/mL, however, 7KC could significantly induce DNA fragmentation in 20 h [44].
Furthermore, we found that 7KC induced oxiapoptophagy in MC3T3-E1 by upregulating NOX4 expression and increasing intracellular ROS, thus reducing the survival rate of MC3T3-E1 cells. The presence of the ROS scavenger NAC could compromise these alterations. ROS are produced in cells under various stresses [45]. However, the excessive production of ROS can destroy the balance between oxidant and antioxidant systems and altered ROS generation can lead to the oxidative-stress-induced OP by promoting lipid peroxidation, reducing antioxidant enzyme expression, inducing osteoblast apoptosis, and inhibiting bone formation [46][47][48][49]. It was initially found that 7KC can increase the expression of NOX4, contributing to the overproduction of ROS in human aortic smooth muscle cells [50]. Consistently, 7KC enhances ROS production, promotes the translocation of NOX components from the cytosol to the cellular membrane, inhibits HO-1 expression, and stimulates lysozyme release in human neutrophils [51]. In human red blood cells, 7KC is reported to activate NOX and induce eryptosis by mediating Rac GTPase and PKCζ [52]. NOX4 is an important source of ROS production and is highly expressed in MC3T3-E1 cells [53]. NOX4 has been involved in the glucocorticoid-induced apoptosis of MC3T3-E1 cells [54]. It was found that simvastatin can protect osteoblasts against H2O2-induced oxidative damage by inhibiting NOX4 expression [55]. In addition, iron overload can stimulate ROS generation, enhance NOX4 expression, and induce apoptosis in MC3T3-E1 cells. NAC can scavenge ROS, inhibit NOX4 expression, and ameliorate iron-overload-induced apoptosis [56].
Oxidative stress can inhibit osteoblast differentiation. Although bone and vascular cells share common processes of osteogenesis and mineralization, reduced bone mineralization (osteoporosis) is associated with increased calcification in humans and mice [57,58]. These paradoxical results in skeletal and vascular niches suggest that different internal signaling pathways orchestrated by tissue-specific microenvironments control the production of mineralization in different ways [59]. Mechanically, this discrepancy might be related to the totally different actions of oxidative stress on RUNX2 expression, which is an important regulator of osteogenic differentiation and mineralization in osteocytes and VSMC [57,60]. Growing evidence demonstrates that oxysterols play a critical role in vascular calcification [6]. Studies have shown that 7KC is also highly involved in PIinduced calcification of vascular smooth muscle cells by inducing lysosomal dysfunction and apoptosis [43,61,62]. A study found that 7KC promotes VSMC cell calcification through lysosomal dysfunction-dependent oxidative stress [43]. Additionally, 7KC may play a key pathogenic role in atherosclerotic plaque calcification development [63]. Our study showed that 7KC inhibited the ALP activity and OPN and RUNX2 expression, thereby inhibiting the osteogenic differentiation of MC3T3-E1 cells. However, these effects could be attenuated by NAC.
Bone homeostasis is maintained by the balance between bone formation and bone resorption. Osteoblasts are differentiated from BMSCs and exhibit critical roles in bone formation. Osteogenic differentiation is complex and orchestrated by various signaling pathways, such as Wnt/β-catenin, TGFβ/BMP, MAPK, and Notch signaling pathways [64]. Early osteogenic biomarkers, such as ALP, RUNX2, and SP7, and late biomarkers, such as OPN and OCN, have been used to evaluate the biological effects of potential candidates [65]. RUNX2 is an important transcription factor mastering osteogenic differentiation that regulates the transcriptional expression of these biomarkers [66,67]. ALP, RUNX2, and OCN build a transcriptional network, governing osteogenesis. In the current study, these biomarkers were purposely employed to evaluate the effects of 7KC on osteogenic differen-tiation. However, their potential mechanisms still need further investigation. Additionally, although our study shows that 7KC can induce MC3T3-E1 oxiapoptophagy and osteogenic differentiation inhibition, these processes are related to the excessive production of ROS. However, the relationship between oxidative stress, apoptosis, autophagy, and osteogenic differentiation was not addressed in detail. More careful studies are required in the future.

Conclusions
7KC, an oxysterol formed by autoxidation, is involved in the pathophysiological processes of many diseases, such as cardiovascular, neuronal, and retinal diseases. In this study, we observed that 7KC significantly enhanced oxidative stress, induced autophagy, and promoted apoptosis in MC3T3-E1 cells. Meanwhile, 7KC attenuated the expression of osteogenic biomarkers, such as ALP, RUNX2, and OPN, indicating inhibitory activity against osteogenic differentiation. It is important to explore the association of oxysterols, particularly 7KC, with the metabolism of osteoblasts/osteoclasts in bone tissues. Oxysterols can be potent intermediates for the synthesis of steroid hormones or bile acids. Additionally, exogenous or endogenous oxidized cholesterol products are easily accessible. The accumulation of these oxysterols, such as 7KC, may exhibit adverse effects. It is essential to understand the underlying regulatory mechanism of oxysterols as they might become potential targets for the therapeutic management of diseases, including OP.

Data Availability Statement:
The data used to support the findings of this study are included within the article.