Propofol induces nuclear localization of Nrf2 under conditions of oxidative stress in cardiac H9c2 cells

Oxidative stress contributes to myocardial ischemia-reperfusion injury, which causes cardiomyocyte death and precipitate life-threatening heart failure. Propofol has been proposed to protect cells or tissues against oxidative stress. However, the mechanisms underlying its beneficial effects are not fully elucidated. In the present study, we employed an in vitro oxidative injury model, in which rat cardiac H9c2 cells were treated with H2O2, and investigated roles of propofol against oxidative stress. Propofol treatment reduced H2O2-induced apoptotic cell death. While H2O2 induced expression of the antioxidant enzyme HO-1, propofol further increased HO-1 mRNA and protein levels. Propofol also promoted nuclear localization of Nrf2 in the presence of H2O2. Knockdown of Nrf2 using siRNA suppressed propofol-inducible Nrf2 and expression of Nrf2-downstream antioxidant enzyme. Knockdown of Nrf2 suppressed the propofol-induced cytoprotection. In addition, Nrf2 overexpression induced nuclear localization of Nrf2 and HO-1 expression. These results suggest that propofol exerts antioxidative effects by inducing nuclear localization of Nrf2 and expression of its downstream enzyme in cardiac cells. Finally, we examined the effect of propofol on cardiomyocytes using myocardial ischemia-reperfusion injury models. The expression level of Nrf2 protein was increased at 15 min after reperfusion in the ischemia-reperfusion and propofol group compared with ischemia-reperfusion group in penumbra region. These results suggest that propofol protects cells or tissues from oxidative stress via Nrf2/HO-1 cascade.


Introduction
Oxidative stress contributes to many pathological conditions, including tissue ischemia, neurological disorders, cancer, hypertension, atherosclerosis, diabetes, idiopathic pulmonary PLOS ONE | https://doi.org/10.1371/journal.pone.0196191 April 24, 2018 1 / 16 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 fibrosis and asthma [1]. Oxidative stress causes an overabundance of oxidants, such as reactive oxygen species (ROS), that are highly reactive and can damage cell components, including carbohydrates, lipids, nucleic acids and proteins, and alter their functions [1]. In the case of cardiac diseases, oxidative stress plays a major role in myocardial ischemia-reperfusion injury that results in cardiac cell death and subsequent heart failure [2]. Propofol (2, 6-diisopropylphenol) is used to sedate patients during surgery [3]. The anesthetic effect of propofol has been attributed to activation of GABA A receptors, and consequent slowing of the channel-closing time. Propofol also acts as a sodium channel blocker [4]. In addition to its anesthetic effects, propofol reportedly protects cells or tissues from oxidative stress [5,6]. The underlying mechanisms of this beneficial effect have not been elucidated. In some cases, however, propofol showed cytotoxic effects [7,8]. Tsuchiya et al. [9] demonstrated that propofol could induce apoptosis in cultured human promyelocytic leukemia HL-60 cells via activation of the cell surface death receptor pathway and the mitochondrial pathway. These discrepancies may be attributed to differences in cell types and/or in experimental paradigms. Whether propofol has beneficial or harmful effects on particular cell types or tissues is clinically important, since propofol is commonly used in surgery, in which the human body receives invasive stress.
Heme oxygenase-1 (HO-1) is an antioxidant enzyme that can be induced by oxidative stress [10]. It catalyzes the rate-limiting step in heme degradation, leading to generation of equimolar amounts of iron ions, biliverdin and CO [10]. Cardiac-specific HO-1 overexpression protects against myocardial ischemia and reperfusion injury [11] and improves cardiac function in an animal model [12]. HO-1 expression is regulated by NF-E2-related factor 2 (Nrf2), a transcription factor that is responsible for the regulation of cellular redox balance [10]. It has been reported that Nrf2 is the principal transcription factor that regulates antioxidant response element-mediated expression of antioxidant enzymes [13,14]. Hao et al. reported that Nrf2 is a key molecule that inhibited endotoxin-induced myocardial toxicity using a mouse model [15]. Although the activation of Nrf2/HO-1 by propofol has been reported in a rat liver transplantation model [5,16], little is known from cardiomyocyte models about the relationship between Nrf2/HO-1 cascades and propofol. In the present study, we employed a H 2 O 2 -induced oxidative stress model to investigate directly the role of propofol against ROS in rat cardiac H9c2 cells.

Lactate dehydrogenase assay
Cytotoxicity to H9c2 cells was measured using a Cytotoxicity LDH Assay kit (Dojindo Molecular Technologies, Japan). H9c2 cells were seeded in 96-well cell culture plates at a density of 2× 10 4 cells per well and cultured in medium containing 100, 500 or 1000 μM H 2 O 2 , or 10, 100 or 1000 μM propofol. The experimental assay was performed following the manufacturer's protocol. LDH catalyzes the conversion of lactate to pyruvate upon reduction of NAD + to NADH/H + ; the added tetrazolium salt is reduced to formazan. The amount of formazan formed correlates with LDH activity. The formazan product was measured with a microtiter plate reader at 490 nm.

TUNEL assay
H9c2 cells were seeded in 4-well cell culture plates at a density of 2× 10 4 cells per well and cultured in medium containing 250 μM H 2 O 2 , or 100 μM propofol. Apoptotic cells were identified by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining using an In Situ Cell Death Detection Kit (Wako) according to the manufacturer's instructions. Briefly, H9c2 cells were fixed with 4% paraformaldehyde for 10 min at room temperature, washed twice with phosphate-buffered saline (PBS), permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate and then rinsed with PBS. Cells were stained with 100 μl terminal deoxynucleotidyl transferase reaction solution at 37˚C for 10 min, and then incubated with 3% H 2 O 2 solution to eliminate intrinsic peroxidase activity. Apoptotically fragmented DNA was detected using horseradish peroxidase antibody and DAB as a substrate.

Generation of construct and transient transfection in H9c2 cells
PCR cloning was performed to amplify Nrf2 cDNA with a primer having an optimal Kozak consensus sequence just before the in-frame first ATG of rat Nrf2 gene. cDNA fragment was inserted into the pcDNA3.1/Myc-His vector (Invitrogen). By using the Lipofectamine 2000 (Invitrogen), H9c2 cells were transfected with a Kozak-Nrf2 construct according to the manufacturer's instructions.

In vivo myocardial ischemia/reperfusion model
All experiments were approved by the Institutional Review Board of Tokai University (#171026). The animals received humane care as required by the institutional guidelines for animal care and treatment in experimental investigations. Male Lewis rats (6-8 weeks) were randomly divided into two groups: 1) ischemia/reperfusion (I/R) group; 2) I/R+propofol group. All rats were anesthetized with 3% sevoflurane inhalation. Rectal temperature was monitored and maintained at 36.0˚C by servo-controlled water pad. An indwelling catheter for infusion by syringe pump of each solution (saline, propofol) was inserted into the tail vein. A left thoracotomy was performed to expose the heart and sevoflurane was reduced to 2% to the end of experiment. The myocardial I/R model was induced by ligation with a 7-0 manofilament suture of the left anterior descending (LAD) coronary artery for 30 minutes followed by reperfusion for 15 min or 90 min. The rats in the I/R group received an intravenous infusion of physiologic saline (3 ml/hr) for 45 min or 120 min. In the I/R+propofol group, 22 mg/kg/hr propofol was started 10 min after LAD occlusion and administered for 35 min or 110 min. At the end of the ischemic period, the suture was released to induce reperfusion of the myocardium for 15 min or 90 min. The following territories of the heart were investigated: 1) Infarction: tissue most vulnerable to damage during a LAD coronary artery occlusion were located at the vascular bed of the LAD. 2) Penumbra: tissue within the vascular bed of the LAD comprised the area at risk.

Statistical analysis
Quantifications were performed from at least three independent experimental groups. Data are presented as mean ± SEM. Statistical analyses were performed using Student's t-test or Welch's t-test for two groups or one-way ANOVA for multiple groups, and significant differences between group means were identified with the Tukey-Kramer test. Statistical significance is indicated as asterisks. Ã P < 0.05, ÃÃ P < 0.01. All n and P values are indicated in figure legends.

Effects of H 2 O 2 and propofol on cell viability
We first determined the dose at which cytotoxicity develops after 24 h H 2 O 2 exposure in H9c2 cells. As shown in Fig 1A, H 2 O 2 caused cell death in a concentration-dependent manner over the tested range (100 to 1000 μM). Propofol has been proposed to protect cells or tissues from oxidative stress [5,6], but reportedly shows cytotoxic effects in cells from the immune system [7,8]. Therefore, we next tested whether propofol was cytotoxic to H9c2 cells. We determined cell cytotoxicity after treatment with propofol (10 to 1000 μM) for 24 h, using phase-contrast microscopy and an LDH assay. Cell viability was not affected by propofol at 10 or 100 μM, but cell death was observed with 1000 μM propofol (Fig 1B and 1C). Because the aim of this study was to investigate protective roles of propofol against ROS, we used 250 μM H 2 O 2 , 100 μM propofol for subsequent experiments.

Propofol inhibits H 2 O 2 -induced H9c2 cell death
To determine the effects of propofol on H 2 O 2 -induced cell death, H9c2 cells were pretreated with propofol (100 μM) for 30 min, and then co-incubated with 250 μM H 2 O 2 for an additional 24 h (Fig 1D). We checked the protective effects of propofol on H 2 O 2 -induced cell death by a trypan blue exclusion test. As shown in Fig 1E and 1F (Fig 1G and 1H).

Propofol and H 2 O 2 synergistically increase HO-1 expression in H9c2 cells
We next examined the expression profiles of oxidative stress-related factors. HO-1 is an important component of the cellular defense mechanism against oxidative stress [10]. Thus, we determined whether propofol induced HO-1 expression in H9c2 cells. As shown in Fig

Propofol upregulate nuclear localization of Nrf2
It has been reported that antioxidant genes including HO-1 are regulated by the transcription factor Nrf2 [10]. We next investigated whether propofol-induced HO-1 expression is Nrf2-dependent in H9c2 cells. The Nrf2 level in total lysates of H9c2 cells was higher in cells pretreated with 100 μM propofol for 30 min and then incubated with 250 μM H 2 O 2 than in cells treated with only 250 μM H 2 O 2 (Fig 2D and 2E, S1C and S1D Fig), although the difference did not reach statistical significance. We noticed that Nrf2 was detected as two bands around 110 kDa. We prepared nuclear extracts and cytoplasmic extracts of H9c2 cells and performed Western blotting. Western blot analysis detected molecular weight of nuclear Nrf2 was higher than that of cytoplasmic Nrf2 (Fig 2F, S1E Fig). It has been reported that under normal conditions, Nrf2 and Keap1 are complexed in the cytoplasm where they are targeted for degradation. On the other hand, Nrf2 is released from Keap1 and translocates to the nucleus [17]. We next investigated whether propofol-induced nuclear Nrf2 expression in H9c2 cells, using Histone H1 as an internal control for nuclear proteins. As shown in Fig 2G and 2H, propofol significantly increased the expression level of nuclear Nrf2 (4.4-fold of control) under conditions of oxidative stress in H9c2 cells (S1F and S1G Fig), whereas cytoplasmic Nrf2 was not changed (Fig 2G  and 2I, S1H and S1I Fig). Notably, combined propofol and H 2 O 2 treatment induced nuclear localization of Nrf2, whereas single propofol treatment did not (Fig 2G and 2H, S1F and S1G Fig). Immunocytochemistry also demonstrated that propofol promoted nuclear localization of Nrf2 in the presence of H 2 O 2 ( Fig 2J). These results suggest that propofol induced HO-1 expression by promoting nuclear localization of Nrf2 under oxidative stress conditions.

Nrf2 overexpression induce nuclear localization of Nrf2 and HO-1 expression
To examine the effects of Nrf2 on expression of antioxidants, we used Kozak-linked Nrf2 construct. Nrf2 construct was transfected in HEK293T cells, and exogenous Nrf2 protein was detected using anti-c-Myc antibody. (Fig 5A, S2A Fig). In Fig 2F, we showed that molecular weight of nuclear Nrf2 was higher than that of cytoplasmic Nrf2 in H9c2 cells. Therefore, we The role of propofol against ROS in H9c2 cells examined whether overexpression of Nrf2 changed localization of Nrf2. As shown in Fig 5B  and 5C, overexpression of Nrf2 upregulated nuclear Nrf2 (N-Nrf2) expression in normal condition (S2B and S2C Fig). Furthermore, propofol increased the expression of nuclear Nrf2 under conditions of oxidative stress in Nrf2 overexpressed H9c2 cells (Fig 5D and 5E, S2D and  S2F Fig). Interestingly, HO-1 was also induced by propofol in Nrf2 overexpressed H9c2 cells (Fig 5D, S2E Fig). These results suggest that overexpression of Nrf2 induced HO-1 expression by promoting nuclear localization of Nrf2 under oxidative stress conditions. The role of propofol against ROS in H9c2 cells

Effects of propofol on myocardial ischemia-reperfusion injury
It is known that propofol protects myocardium against myocardial ischemia-reperfusion (I/R) injury in rat heart model [19]. However, the mechanisms underlying its cardioprotective role remain elusive. Our in vitro studies showed that propofol-induced Nrf2 is critical for cytoprotection against oxidative stress. Therefore, we examined the expression level of Nrf2 downstream enzyme, HO-1 in I/R injury model. The myocardial I/R model was induced by ligation of the left anterior descending (LAD) coronary artery for 30 minutes followed by reperfusion for 15 min or 90 min (Fig 6A and 6B). In the I/R+propofol group, 22mg/kg/hr propofol was administered for 35 min or 110 min (Fig 6A). HO-1 mRNA level was increased after 15 min on reperfusion in the I/R+propofol group compared with I/R group in penumbra and infarction region, although the difference did not reach statistical significance (Fig 6C). HO-1 mRNA level was not increased after 90 min on reperfusion in the I/R+propofol group in The role of propofol against ROS in H9c2 cells The role of propofol against ROS in H9c2 cells penumbra and infarction region. We focused on the early stage since HO-1 mRNA tended to be higher at 15 min after reperfusion in propofol-treated rats. We performed Western blotting using ischemia/reperfusion (I/R) group rats and I/R+propofol group rats at 15 min after reperfusion. The Nrf2 level in penumbra region was higher in the I/R+propofol group compared with I/R group in penumbra region (Fig 6D, S3 Fig 3.3-fold of I/R group). In infarction region, Nrf2 expression level was not changed between I/R+propofol group and I/R group (Fig 6D, S3  Fig). These results suggest that propofol increased the Nrf2 and its downstream enzyme especially in penumbra region at the early stage of reperfusion.

Discussion
Propofol has been proposed to contribute to the protection of cells or tissue against oxidative stress [5,6]. However, the underlying mechanism of this beneficial effect is not clear. It is widely accepted that mitochondria play a key role in the development of oxidative stress, and the major endogenous sources of ROS are localized to mitochondria. In the present study, we used exogenous H 2 O 2 to investigate directly the role of propofol against ROS. As shown in Fig  1, propofol inhibits H 2 O 2 -induced apoptosis of H9c2 cells. Antioxidants such as HO-1 and Nqo-1 are important components of the cellular defense mechanism against oxidative stress [10]. We showed that propofol and H 2 O 2 co-treatment increased the expression of HO-1 by promoting Nrf2 nuclear localization in H9c2 cells. In other words, propofol has protective effects only if cells are under oxidative stress. Interestingly, we found that the mRNA expression level of Nrf2 was increased by propofol in the presence of H 2 O 2 (S1J Fig). Propofol appears to regulate Nrf2 expression at the transcription level; however, propofol treatment alone did not increase Nrf2 mRNA expression. These results indicate that propofol and H 2 O 2 synergistically increase Nrf2 mRNA expression in H9c2 cells. Identification of the molecular mechanisms that regulate Nrf2 transcription will be the next step.
Interestingly, propofol (100 μM) did not protect 1000 μM H 2 O 2 -treated H9c2 cells from cell death (data not shown), suggesting that extreme conditions may preclude the protective effects of propofol. We also observed that 1000 μM propofol, but not 100 μM, caused cell death in H9c2 cells (Fig 1B and 1C). It has been reported that excessive propofol treatment for a prolonged period causes injury to multiple cell types [9,18]. Indeed, abuse of propofol treatment causes severe complications in patients with critical illness and is called propofol infusion syndrome [20]. The mechanism of cellular cytotoxicity caused by propofol overdose is still unclear. Our aim was to investigate protective roles of propofol against ROS, and we therefore used 100 μM propofol in this study.
Nrf2 activates transcription of many cytoprotective enzymes [21], and HO-1, Nqo-1, gluthathione S-transferase and superoxide dismutase are representative antioxidant enzymes whose expression is regulated by Nrf2 [10,13,14,21]. The activation mechanism of Nrf2 has been studied extensively. Hao et al. reported that polyphenolic compound, resveratrol alleviates endotoxin-induced myocardial toxicity via the Nrf2 transcription factor [15]. In addition, Ryu et al. reported that 7, 8-dihydroxyflavone protects human keratinocytes against oxidative stress-induced cell damage via the ERK and PI3K/Akt-mediated Nrf2/HO-1 signaling pathways [22]. To examine the importance of Nrf2 induced by propofol, we disrupted Nrf2 expression using siRNA. Nrf2, Nqo-1 and HO-1 mRNA levels were higher in cells treated with H 2 O 2 or H 2 O 2 /propofol than in non-treated cells due to incomplete knockdown by Nrf2 siRNA. Nonetheless, Nqo-1 mRNA was significantly reduced by Nrf2 siRNA (Fig 3), but not by siRNA against HO-1 (data not shown). In addition, propofol did not reduce the H 2 O 2 -induced cytotoxicity of H9c2 cells transfected with Nrf2 siRNA (Fig 4). When we conducted the same experiment using HO-1 or Nqo-1 siRNA, propofol reduced the H 2 O 2 -induced cytotoxicity of H9c2 cells to the same extent as scramble siRNA (Fig 4C and 4D). In addition, to examine the effects of Nrf2 on expression of antioxidants and cytoprotection, we used Kozak-linked Nrf2 construct. Exogenous Nrf2 protein was detected using anti-c-Myc antibody. (Fig 5). However, Western blot analysis showed that a band of Nrf2 protein in cell lysates was weak. It has been reported that under normal conditions, Nrf2 and Keap1 are complexed in the cytoplasm where they are targeted for degradation [10]. The exogenous Nrf2 protein might be degraded in our in vitro assay system. Indeed, propofol did not reduced the H 2 O 2 -induced cytotoxicity of H9c2 cells transfected with Nrf2 construct (H 2 O 2 , 33.4% ± 3.0%; H 2 O 2 +PR, 32.0% ± 2.7%) (S2G Fig). However, overexpression of Nrf2 upregulated nuclear localization of Nrf2 even a band of Nrf2 protein in cell lysates was weak (Fig 5). Furthermore, HO-1 was also induced by propofol in Nrf2 overexpressed H9c2 cells (Fig 5). These results suggested that overexpression of Nrf2 induced HO-1 expression by promoting nuclear localization of Nrf2 under oxidative stress conditions. These results suggest that propofol-induced Nrf2 expression is critical for antioxidant expression.
It is known that propofol protects myocardium against myocardial ischemia-reperfusion (I/R) injury in the rat heart model [19]. However, the mechanisms underlying its cardioprotective role remain elusive. We examined expression levels of Nrf2 in ischemia-reperfusion injury model. Nrf2 was increased after 15 min on reperfusion in the I/R+propofol group compared with I/R group in penumbra and infarction region (Fig 6D). In our study, the myocardial I/R model was induced by ligation of the LAD coronary artery for 30 minutes followed by reperfusion for 15 min or 90 min. HO-1 and Nrf2 were increased after 15 min on reperfusion in propofol-treated rats (Fig 6C and 6D). These results suggest that propofol-induced Nrf2/HO-1 expression is increased in the early stage of reperfusion at penumbra region. Wang et al. reported that administration of propofol ameliorated the cardiac function of rats at 2 h after reperfusion [23]. It is presumed that the expression of Nrf2 and its downstream enzyme including HO-1 are elevated in the early reperfusion stage and contribute to restoration of cardiac function.
In summary, we showed that propofol increased cell survival and reduced H 2 O 2induced apoptosis in rat cardiac H9c2 cells. We also showed that propofol promotes nuclear localization of Nrf2 under conditions of oxidative stress. These results suggest that propofol enhances nuclear Nrf2 accumulation and the expression of its downstream enzyme(s), thereby protecting cardiac H9c2 cells from oxidative stress-induced cell death. Further characterization of propofol and investigation of the mechanism by which it regulates gene expression should provide new insights into its crucial role under oxidative stress conditions.