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Propofol protects human keratinocytes from oxidative stress via autophagy expression

Background The skin consists of tightly connected keratinocytes, and prevents extensive water loss while simultaneously protecting against the entry of microbial pathogens. Excessive cellular levels of reactive oxygen species can induce cell apoptosis and also damage skin integrity. Propofol (2,6-diisopropylphenol) has antioxidant properties. In this study, we investigated how propofol influences intracellular autophagy and apoptotic cell death induced by oxidative stress in human keratinocytes. Method The following groups were used for experimentation: control, cells were incubated under normoxia (5% CO2, 21% O2, and 74% N2) without propofol; hydrogen peroxide (H2O2), cells were exposed to H2O2 (300 µM) for 2 h; propofol preconditioning (PPC)/H2O2, cells pretreated with propofol (100 µM) for 2 h were exposed to H2O2; and 3-methyladenine (3-MA)/PPC/H2O2, cells pretreated with 3-MA (1 mM) for 1 h and propofol were exposed to H2O2. Cell viability, apoptosis, and migration capability were evaluated. Relation to autophagy was detected by western blot analysis. Results Cell viability decreased significantly in the H2O2 group compared to that in the control group and was improved by propofol preconditioning. Propofol preconditioning effectively decreased H2O2-induced cell apoptosis and increased cell migration. However, pretreatment with 3-MA inhibited the protective effect of propofol on cell apoptosis. Autophagy was activated in the PPC/H2O2 group compared to that in the H2O2 group as demonstrated by western blot analysis and autophagosome staining. Conclusion The results suggest that propofol preconditioning induces an endogenous cellular protective effect in human keratinocytes against oxidative stress through the activation of signaling pathways related to autophagy.


INTRODUCTION
Human skin is composed of tightly connected keratinocytes in the stratum granulosum and terminally differentiated keratinocytes in the stratum corneum. This intricate constitution comprises an effective barrier that resists extensive water loss while simultaneously protecting the cell from the entry of microbial pathogens and allergens [1]. Surgical damage can disrupt this barrier, and a fair number of patients have wound healing abnormalities [2]. In addition, non-healing wounds can deteriorate chronic ulcer or cause wound-related secondary infections, and these conditions make it harder to recover from disease. At worst, chronic wound tissue can develop into cancer due to persistent inflammation [3].
Propofol (2,6-diisopropylphenol) is a widely used agent for the induction and maintenance of anesthesia during surgical procedures. It is also used as a sedative for intensive care unit (ICU) patients. Propofol has structural similarity to the endogenous antioxidant vitamin E and shows antioxidant activity [4]. Propofol has a protective effect on hydrogen peroxide (H 2 O 2 )-induced apoptosis in cardiac cells [5] and in myocardial ischemia and reperfusion injury in rats [6]. However, the effect of propofol on human keratinocytes under oxidative stress is yet to be fully elucidated. In this study, we investigated how propofol influences intracellular autophagy and apoptotic cell death induced by oxidative stress in human keratinocytes. The antibodies used in the study were as follows: Bak (1:1000), caspase-9 (1:1000), caspase-3 (1:1000), and poly (ADP-ribose) polymerase (PARP; 1:1,000) (Abcam, Cambridge, MA, USA). The medium was changed once every 3 days.

Oxidative stress of cultured human keratinocytes and drug treatment
The propofol solution was prepared by dissolving propofol in DMSO, which was kept frozen at -4°C until use. The stock was diluted to the appropriate concentration with DMEM when needed. Prior to propofol treatment, cells were grown to approximately 75% confluence and then exposed to propofol at various concentrations (0, 3, 30, 100, and 300 μM) for 2 h. The following four groups were used for experimentation: control, cells were incubated at 37°C in a humidified atmosphere with 5% CO 2 without propofol treatment;. H 2 O 2 , cells were exposed to 300 μM hydrogen peroxide (H2O2) for 2 h; PPC/H2O2, cells were pretreated with 100 μM propofol for 2 h and exposed to 300 μM H 2 O 2 for 2 h; and 3-MA/PPC/H 2 O 2 , cells were pretreated with 1 mM 3-MA and 100 μM propofol for 1 h and 2 h, respectively. After treatment of 3-MA and propofol, cells were exposed to 300 μM H 2 O 2 for 2 h (Fig. 1).

Cell viability assay
The cell viability was determined using an MTT assay.

Western blot analysis
The cells (1.     h of incubation in growth medium at 37°C, the new growth and migration into the cell-free zone was considered as the process of in vitro healing. Images of the relatively healed monolayer area were obtained and the pixel counts were analyzed using NIH ImageJ software.

Propofol improved cell viability under oxidative stress
The effect of propofol was investigated over a wide lower cell viabilities than the control group did (Fig. 3). Moreover, flow cytometry was used to detect the percen-tage of apoptotic cells (Fig. 5). Compared to that in the control group and PPC/H 2 O 2 group, the number of   Fig. 6. Effects of propofol on the expression of Bak, caspase-9, caspase-3, and poly (ADP-ribose) polymerase (PARP) in human keratinocytes. Western blot analysis. In the PPC/H2O2 group, cellular expression of caspase-9, caspase-3, and PARP was elevated while that of Bak was down-regulated.  Fig. 6).

Propofol led to the activation of autophagy in human keratinocytes
A significant accumulation of autophagy-specific staining of MDC was observed around the nuclei in cells of the PPC/H 2 O 2 group (Fig. 7). Similar results were observed upon AO staining; red fluorescent spots were

Propofol stimulated cell migration in the woundhealing assay
An in vitro wound-healing assay was performed to determine the migratory capability of human keratinocytes. Wound healing was monitored at 0 and 24 h. The PPC/H2O2 group treated with propofol showed increased wound healing capability (Fig. 9).

DISCUSSION
Skin cells are equipped with an elaborate system that maintains the balance between oxidative stress and antioxidant defense. This system can protect the cells from oxidative stress damage [7]. The excessive cellular levels of reactive oxygen species, such as singlet oxygen, superoxide anions, and H 2 O 2 have been linked to apoptosis. This oxidative stress can induce the damage of cellular proteins, lipids, and polysaccharides, and may also indirectly cause structural damage to DNA [8][9][10].
The biology of keratinocytes is divided into two pathways associated with cell death: apoptosis and differentiation. The pathway that leads to terminal differentiation is known as cornification, which has several steps.
Other authors have described this process as a specialized form of programmed cell death, in which the cells are not removed by phagocytosis like the apoptotic process, and it is used to maintain homeostasis in the skin [11].
Apoptosis is a complex process of purposeful suicide by a cell in a multi-cellular organism. It is one of the main types of programmed cell death and involves biochemical events leading to a characteristic cell morphology and death [12]. Autophagy, an evolutionary conserved process of "self-eating," occurs at a basal level in most cells and contributes to the turnover of long-lived proteins and organelles to maintain intracellular homeostasis [13]. Accordingly, apoptosis and autophagy constitute the two main pathways through which superfluous, damaged, or aged cells and organelles are eliminated.
Previous reports have shown that the cellular protective mechanism of propofol may partly result from its ability to act as a free radical scavenger [14][15][16]. The aim of this study was to determine the protective effect of propofol on human keratinocytes against oxidative stress and to investigate whether autophagy is associated with this protective mechanism. From this study, we present four principal findings. Second, propofol preconditioning exerted an antiapoptotic effect on human keratinocytes exposed to oxidative stress. In western blot analysis, we found that propofol preconditioning increased the cellular expression of caspase-9, caspase-3, and PARP, and decreased Bak expression. Among the factors associated with apoptosis, caspase-9, caspase-3, and PARP play an important role in the caspase-dependent pathway, and the pro-apoptotic protein Bak is related to the mitochondria-dependent apoptotic pathway [17][18][19].
Third, the anti-apoptotic effect of propofol preconditioning in human keratinocytes was probably due to the induction of intracellular autophagy. 3-MA is an intracellular autophagic pathway inhibitor that increases cellular apoptosis, and is suggested to play an important role in intracellular autophagy in the anti-apoptotic effect of propofol preconditioning. In the autophagy-specific staining with MDC and AO, the PPC/H 2 O 2 group showed greater expression of the autophagosome than the other groups did.
Finally, an in vitro wound-healing assay was performed to determine the migratory ability of human keratinocytes.
The PPC/H 2 O 2 group showed increased migratory capability. Migration of keratinocytes is an essential process in wound healing [20].
Based on these results, although the current study could not demonstrate the protection by propofol preconditioning in vivo, we suggest that propofol preconditioning induced an endogenous cellular protective effect in human keratinocytes against oxidative stress through the activation of signaling pathways related to autophagy.
Finally, we believe that our results would contribute to the body of knowledge regarding the relationship between wound healing and oxidative stress.
NOTES: Thesis for the degree of Doctor of Philosophy.
There are no financial or other issues that might lead to conflict of interest.