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Effects of propofol-induced autophagy against oxidative stress in human osteoblasts

Background Oxidative stress occurs during the aging process and other conditions such as bone fracture, bone diseases, and osteoporosis, but the role of oxidative stress in bone remodeling is unknown. Propofol exerts antioxidant effects, but the mechanisms of propofol preconditioning on oxidative stress have not been fully explained. Therefore, the aim of this study was to evaluate the protective effects of propofol against H2O2-induced oxidative stress on a human fetal osteoblast (hFOB) cell line via activation of autophagy. Methods Cells were randomly divided into the following groups: control cells were incubated in normoxia (5% CO2, 21% O2, and 74% N2) without propofol. Hydrogen peroxide (H2O2) group cells were exposed to H2O2 (200 µM) for 2 h, propofol preconditioning (PPC)/H2O2 group cells were pretreated with propofol then exposed to H2O2, 3-methyladenine (3-MA)/PPC/H2O2 cells were pretreated with 3-MA (1 mM) and propofol, then were exposed to H2O2. Cell viability and apoptosis were evaluated. Osteoblast maturation was determined by assaying bone nodular mineralization. Expression levels of bone related proteins were determined by western blot. Results Cell viability and bone nodular mineralization were decreased significantly by H2O2, and this effect was rescued by propofol preconditioning. Propofol preconditioning effectively decreased H2O2-induced hFOB cell apoptosis. However, pretreatment with 3-MA inhibited the protective effect of propofol. In western blot analysis, propofol preconditioning increased protein levels of collagen type I, BMP-2, osterix, and TGF-β1. Conclusions This study suggests that propofol preconditioning has a protective effect on H2O2-induced hFOB cell death, which is mediated by autophagy activation.


INTRODUCTION
Human bone is a highly active organ that maintains its homeostasis through an exquisite balance between bone formation by osteoblasts and bone resorption by osteoclasts. The energetic balance between these two cell types results in bone remodeling [1][2][3][4]. Imbalance of free radical production and redox mechanisms can result in oxidative stress, which damages cellular components and their functions. Oxidative stress is responsible for the aging process and many diseases such as bone fracture, bone diseases, and osteoporosis [5]. The role of free radicals and oxidative stress in bone remodeling is unknown [6].
There is a clear difference between reactive oxygen species (ROS) required for basic cellular mechanisms and excessive ROS production that might cause oxidative stress and contribute to the pathogenesis of major diseases such as diabetes, neurodegeneration, and cancer [7].
Among the various ROS, hydrogen peroxide (H 2 O 2 ) is perhaps the most ubiquitous of these species, which is found at measurable levels in all animal tissues. H2O2 is very stable and can reach molecular targets distant from its site of generation. Because H 2 O 2 is a small, uncharged molecule, it easily crosses cell membranes and localizes in multiple subcellular compartments [8]. The effects of H 2 O 2 are concentration-dependent and range from physiological signaling in cell proliferation, migration, survival, differentiation, and gene expression [9][10][11][12] to overt cell death [13][14][15].
Propofol (2,6-diisopropylphenol) is an intravenous sedative-hypnotic agent used for general anesthesia and sedation of patients in intensive care unit [16,17]. Propofol has a similar structure to the endogenous antioxidant vitamin E and exhibits antioxidant activities [18,19].
In other studies, administration of anesthetic agents was found to result in pharmacologic preconditioning against oxidative injury [20,21].
Autophagy is a conserved cellular catabolic process that can engulf cytoplasmic components and degrade them through the lysosomal pathway, thus, they can then be recycled. Generally, autophagy is thought to be induced under stress conditions [22][23][24]. However, whether the induction of autophagy contributes to cell survival or cell death remains elusive. Oxidative injury is one of the most common causes of bone remodeling inhibition. Therefore, a sustainable drug is needed to identify better, safer anabolic agents with low cytotoxicity that act by either increasing osteoblast proliferation or differentiation to enhance bone formation [25]. Several lines of evidence have shown that anesthesia drugs may have a beneficial effect on bone loss and fracture outcomes [26]. On the basis of this, it was hypothesized that the antioxidant properties of propofol may protect human fetal osteoblast (hFOB) cells against oxidative injury caused by H 2 O 2 . Therefore, the aim of this study was to evaluate the protective effects of propofol-

Propofol treatment
The propofol stock (56 mM) was kept at room temperature until use and diluted to the proper concentration with DMEM/F-12 when needed. Prior to propofol treatment, cells were grown to about 75% confluence and then   The experimental paradigm as just described is visualized in Fig. 1.

Cell viability assay
The cell viability of hFOB cells was determined using an MTT assay. Cells were cultured in 96-well plates (4 The cell viability was measured by an ELISA reader (Tecan, Männedorf, Switzerland) by determining the wells' absorbance at 620 nm.

Statistical analysis
The data are expressed as mean ± SE. The statistical significance between groups was anlalyzed using oneway analysis of variance (ANOVA) and a Dunnett's comparison. Probability (p) values less than 0.05 were considered statistically significant.

Effect of propofol treatment on cell proliferation
The effect of propofol on hFOB cells was investigated over a wide concentration range. We pretreated hFOB cells with various doses of propofol, exposed the cells to oxidative stress, and then measured cell viability by an MTT assay. The viability of propofol-treated hFOB cells increased in a dose-dependent manner. Propofol and 3-MA alone did not show any significant toxic effects on hFOB cells ( Fig. 2A, 2B). Incubation with 200 μM H2O2 markedly decreased cell viability, which was significantly attenuated when treated with propofol, suggesting that propofol abated H 2 O 2 -induced oxidative stress in osteoblasts (Fig. 3).

Propofol protected against H 2 O 2 -induced apoptosis in hFOB cells
The effect of propofol on apoptosis was examined by Hoechst 33342 staining of hFOB cells exposed to control,

Effect of propofol treatment on autophagy activation
Prominent accumulation of autophagy-specific MDC staining was observed around the nuclei in the PPC/H 2 O 2 group (Fig. 6). Similarly, after AO staining, red fluorescent spots appeared in PPC/H2O2 group hFOB cells, while the control and 3-MA groups showed mainly green cytoplasmic fluorescence (Fig. 7).

Effect of propofol preconditioning on bone nodular mineralization
Abundant bone nodular mineralization occurred in the control group, and propofol preconditioning had no influence on this. Cells in the PPC/H 2 O 2 group showed higher bone nodular mineralization compared to the control group and bone nodular mineralization increased gradually with increasing propofol preconditioning (Fig. 8).

Effect of Propofol preconditioning on collagen type I, BMP-2, osterix, and TGF-β 1
Propofol preconditioning was found to increase the expression of bone-related proteins such as collagen type I, BMP-2, osterix, and TGF-β1 (Fig. 9). These results suggest that pretreatment with propofol induces the expression of collagen type I, BMP-2, osterix, and TGF-β

DISCUSSION
We demonstrated that propofol preconditioning increased the proliferation, differentiation, and maturation of osteoblasts during oxidative injury in our study. The healing process is very important to recover from fractures, and delayed healing after bone implants can cause premature loosening or failure. In our cell viability analysis, there is a distinction between our study and a previous study, which reported an inhibitory effect of non-steroidal anti-inflammatory drugs on fracture healing [27].
In this study, we demonstrated that propofol preconditioning has positive effects on the expression of collagen type I, BMP-2, osterix, and TGF-β1. BMP-2 and TGF-β1 play a crucial role in inducing osteoblast differentiation and bone formation at the preosteoblast stage [28,29].
These proteins play an important role in the differentiation of osteoblast progenitor cells, with significant upregulation observed in both matrix synthesis and mineralization [30][31][32]. Collagen type I plays a crucial role in cell adhesion, proliferation, and differentiation of osteoblasts [33]. Osterix is a transcription factor that regulates important osteoblast genes such as collagen type I, osteocalcin, and osteopontin [34]. Chen et al. [35] suggested that ROS play a key role in the regulation of autophagy. In this study, propofol preconditioning not only decreased H2O2-induced oxidative stress, but also activated autophagy. Many studies have focused on oxidative injury in cells, but agents that could effectively protect against oxidative injury remain unidentified. The relationship between apoptosis and autophagy is complex. Autophagy represents a stress adaptation for inhibition of cell death, however in other cellular settings, autophagy represents an alternative pathway to autophagic cell death [36,37].
This study shows that propofol pretreatment increases the osteoblast proliferation rate. No functional studies were performed to investigate the effects of propofol on the bone healing process. Therefore, although the findings of this study are limited to an in vitro interpretation, we suggest that propofol may have a beneficial effect in the recovery from bone stress injury.