Propofol Attenuates Hypoxia-Induced Inflammation in BV2 Microglia by Inhibiting Oxidative Stress and NF-κB/Hif-1α Signaling

Hypoxia-induced neuroinflammation typically causes neurological damage and can occur during stroke, neonatal hypoxic-ischemic encephalopathy, and other diseases. Propofol is widely used as an intravenous anesthetic. Studies have shown that propofol has antineuroinflammatory effect. However, the underlying mechanism remains to be fully elucidated. Thus, we aimed to investigate the beneficial effects of propofol against hypoxia-induced neuroinflammation and elucidated its potential cellular and biochemical mechanisms of action. In this study, we chose cobalt chloride (CoCl2) to establish a hypoxic model. We found that propofol decreased hypoxia-induced proinflammatory cytokines (TNFα, IL-1β, and IL-6) in BV2 microglia, significantly suppressed the excessive production of reactive oxygen species, and increased the total antioxidant capacity and superoxide dismutase activity. Furthermore, propofol attenuated the hypoxia-induced decrease in mitochondrial membrane potential andy 2 strongly inhibited protein expression of nuclear factor-kappa B (NF-κB) subunit p65 and hypoxia inducible factor-1α (Hif-1α) in hypoxic BV2 cells. To investigate the role of NF-κB p65, specific small interfering RNA (siRNA) against NF-κB p65 were transfected into BV2 cells, followed by exposure to hypoxia for 24 h. Hypoxia-induced Hif-1α production was downregulated after NF-κB p65 silencing. Further, propofol suppressed Hif-1α expression by inhibiting the upregulation of NF-κB p65 after exposure to hypoxia in BV2 microglia. In summary, propofol attenuates hypoxia-induced neuroinflammation, at least in part by inhibiting oxidative stress and NF-κB/Hif-1α signaling.


Introduction
Microglia are the primary neuroimmune cells important for surveillance and defense, in addition to maintaining homeostasis, being the first sensors of pathophysiological changes, and triggering subsequent cascade reactions [1][2][3]. Microglia undergo a remarkable transformation into "activated microglia" during brain injury and disease, when numerous genes are switched on. M1-activated microglia mainly secrete proinflammatory cytokines (TNF-α, IL-1β, and IL-6), which consequently promote the development of several central nervous system (CNS) disorders. However, M2-activated microglia primarily secrete anti-inflammatory cytokines, which can facilitate tissue reconstruction [4,5]. In a hypoxic condition, Hif-1α is stabilized and activates the transcription of genes associated with proinflammation [6,7]. Moreover, hypoxia impairs mitochondrial function resulting in increased production of reactive oxygen species (ROS), decreased mitochondrial membrane potential, and decreased ATP production [8,9]. Considering the key role of neuroinflammation in hypoxia-induced nerve damage, neuroinflammation induced by activated microglia (especially M1 polarization) is an important regulator of hypoxia-induced CNS damage.
As a commonly used intravenous anesthetic, propofol has many other functions [10,11]. In our previous study, we found that propofol can prevent oxidative stress and attenuate mitochondrial dysfunction during focal cerebral ischemia-reperfusion injury [12,13]. Further, propofol has an anti-inflammatory effect as it inhibits NF-κB activation in mice with allergic asthma [14]. NF-κB subunit p65 has been reported to induce basal level expression of Hif-1α mRNA and protein [15][16][17]. However, the mechanism by which propofol attenuates hypoxia-induced neuroinflammation in activated microglia is relatively unclear. Therefore, we designed this study to investigate the protective effect of propofol on hypoxia-induced neuroinflammation associated with microglia and whether this occurs through the inhibition of oxidative stress and NF-κB/Hif-1α signaling.

Materials and Methods
2.1. Microglial Cell Culture. Since BV2 microglia originate from mouse brains, they share several phenotypic characteristics with primary microglia. Thus, we chose these cells as the model for the current study. The cells were obtained from the National Infrastructure of Cell Line Resource (China). Microglia were grown according to the recommended conditions described by the cell bank. The medium contained high-glucose Dulbecco's modified Eagle's medium (DMEM) (Corning, USA) with 10% fetal bovine serum (FBS) (Corning, USA) and 1% penicillin-streptomycin. The cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO 2 . After growing them to 80% confluence, cells were subcultured two or three times every week. Microglia were seeded in a 6-well plate for 24 h for subsequent experiments, and the inoculation density was 0:5 × 10 6 cells/ml.

Cell Treatment.
In this study, 300 μM CoCl 2 was used to establish hypoxia. According to the preliminary results of cell viability, the 50% inhibitive concentration (IC50) of CoCl 2 was 300 μM at 24 h. Moreover, Hif-1α protein was expressed stably. CoCl 2 was dissolved in distilled water to 300 mM. CoCl 2 solution was filtered with a 0.22 mm Millipore membrane and stored at -20°C. Serum-free medium was used to dilute CoCl 2 for cell treatment. Propofol was used to pretreat cells for 3 h before CoCl 2 treatment. Following propofol treatment, the cells were washed twice with PBS.
2.3. Real-Time Quantitative PCR (qPCR). Microglia were treated as indicated, and total RNA was collected with a TRIzol reagent (Sigma, America). cDNA was prepared using 1 μg of total RNA as the template with a reverse transcription kit (Takara, Japan). The reverse transcription apparatus was set up according to the kit instructions. Real-time PCR analysis was performed using an ABI Prism 7500 fast real-time PCR System (Applied Biosystems, America) with SYBR green. ΔΔCT values were used for analysis. qPCR was set up with a SYBR green PCR master mix (Roche, Switzerland), specific primers, cDNA, and double-distilled water. The conditions for PCR cycles were as follows: predenaturation (94°C for 2 min), denaturation (94°C for 15 s), annealing, and extension (60°C for 30 s). β-Actin was used as a housekeeper gene control, and untreated cells were used as a control to normalize the relative amounts of target gene expression. qPCR primer sequences are shown in Table 1. 2.4. Enzyme-Linked Immunosorbent Assay (ELISA). Microglia were treated as indicated, and cell supernatants were used to measure the concentration of TNF-α, IL-1β, and IL-6 with ELISA kits (BOSTER, China), according to the manufacturer's instructions. The absorbance, using an iMark microplate reader (SpectraMax M3, Molecular Devices, America), was measured at a wavelength of 450 nm. The cytokine concentrations were determined based on reference standard curves.

Western
Blotting. After treating microglia as indicated, cells were lysed with RIPA buffer containing a protease inhibitor and phosphatase inhibitor (BOSTER, China) at a ratio of 100 : 1 : 1. After the cells were completely lysed, the supernatant was taken, and the protein concentration was measured using a BCA protein quantitative kit (BOSTER, China). The same amount of protein was separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Roche, Switzerland). Tween 20 (BOSTER, China) in Tris-buffered saline (BOSTER, China), containing 5% skim milk powder, was used to block the membrane for 2 h. Primary antibodies against NF-κB p65 (1 : 1400, ab16502, Abcam), Hif-1α (1 : 1000, 14179, Cell Signaling Technology), and GAPDH (1 : 1000, TA-08, ZSGB-Bio) were then incubated with PVDF membranes at 4°C overnight. Next, secondary antibodies (peroxidase-labeled) were incubated with the blots at 25°C for 1 h. The protein bands were observed using the enhanced chemiluminescence (ECL) system (GE Healthcare, UK), and relative protein expression was quantified using ImageJ software.

Reactive Oxygen Species Assay Using Flow Cytometry.
Drug intervention was completed as expected, and cells were stained with DCFH-DA (10 μM) for 20 min at 37°C in the dark. Following this, the cells were washed twice with PBS. Cells were removed from the plate surface and transferred to polypropylene FACS tubes with 500 μl PBS. Finally, a MoFlo XDP flow cytometer (Beckman, USA) was used for measurements and FlowJo software for data analysis.
BioMed Research International were then washed twice with PBS. Subsequently, the fluorescence intensity was measured by confocal laser scanning microscopy at an excitation wavelength of 561 nm, and data were analyzed using ImageJ software.
2.8. NF-κB p65 siRNA Transfections. siRNA targeted at NF-κB p65 (Sangon Biotech, China) was used to knock down NF-κB p65. The target sequence was 5′-CAACCATGGCT GAAGGAAA-3′. First, 100 pmol NF-κB p65 siRNA duplex was diluted into 250 μl Opti-MEM medium. Second, 5 μl lipofectamine 2000 (Lipo2000) transfection reagent (Thermo Fisher Scientific, America) was diluted with 250 μl Opti-MEM medium and incubated for 5 min at room temperature in a separate tube. Third, the aforementioned solutions were mixed gently and incubated for 30 min at 25°C to form a transfection complex. Next, 70% confluent BV2 cells to be used in the study were washed thrice with PBS. Following this, 1500 μl Opti-MEM medium and 500 μl transfection complex were added. The cells were incubated for 24 h at 37°C. Western blotting was performed to detect NF-κB p65 protein levels.
2.9. Statistical Analysis. Statistical analyses were performed using IBM SPSS Statistics 24 software. All data presented are representative of at least three independent experiments. The data are shown as the mean ± standard errors of the mean ðS:E:M:Þ. Independent Student's t-test was used for comparisons between two groups, and one-way analysis of variance (ANOVA) with the SNK post hoc test was used to compare multiple groups. The result was considered to be statistically significant when the P value was less than 0.05.

CoCl 2 Exposure Induces the Expression of Proinflammatory Cytokines in BV2 Cells.
To observe the effect of CoCl 2 treatment time on inflammatory responses in microglia, BV2 cells were treated with CoCl 2 (300 μM) for 3, 6, 12, and 24 h. TNF-α, IL-1β, IL-6, and iNOS mRNA expression was detected by qPCR. As shown in Figure 1, TNF-α, IL-1β, IL-6, and iNOS mRNA expression was significantly increased in CoCl 2 -treated cells at 12 and 24 h compared to that in control cells ( * * P < 0:01). Further, compared to that in control cells, IL-1β and iNOS expression   BioMed Research International was significantly increased in cells treated with CoCl 2 at 6 h ( * P < 0:05). Based on the results, we infer that the expression of cytokines in BV2 cells was remarkably increased with prolonged CoCl 2 incubation times.

Propofol Ameliorates the Decrease in Mitochondrial
Membrane Potential in CoCl 2 -Treated Microglia. To determine whether propofol affects the CoCl 2 -induced decrease in mitochondrial membrane potential in BV2 cells, we pretreated cells with propofol for 3 h before CoCl 2 stimulation for 24 h. We then measured mitochondrial membrane potential by confocal laser scanning microscopy. As shown in Figure 4, CoCl 2 induced a decrease in mitochondrial membrane potential in BV2 cells compared to control cells, whereas propofol attenuated this decrease.

Discussion
Propofol, widely used as a short-acting intravenous anesthetic, has chemical properties similar to those of tocopherol, a phenolic free radical scavenger. Moreover, it is lipophilic and can quickly enter cells and subcellular membrane compartments. Thus, propofol has many other functions besides   7 BioMed Research International its anesthetic effect [18,19]. In our previous study, we found that propofol can prevent oxidative stress and attenuate mitochondrial dysfunction upon focal cerebral ischemiareperfusion injury [12,13]. Furthermore, it has an antineuroinflammatory effect and can also inhibit NF-κB activation [20]. NF-κB subunit p65 was reported to contribute to basal levels of Hif-1α mRNA and protein expression [15][16][17]21]. During hypoxia, Hif-1α is stabilized and activates the transcription of genes associated with proinflammation [6,7]. However, the protective effect of propofol against hypoxiainduced neuroinflammation has rarely been reported. In this study, we found that propofol has a protective effect against hypoxia-induced neuroinflammation, at least in part by inhibiting oxidative stress and NF-κB/Hif-1α signaling.
It is known that hypoxia impairs mitochondrial function resulting in ROS production, decreased mitochondrial membrane potential, and metabolic changes [8,9]. ROS are proinflammatory factors which initiate inflammatory cascade reactions and are the main signaling molecules that regulate macrophage phagocytosis and killing [8,36]. ROS oxidizes Fe 2+ to Fe 3+ , an important cofactor that inhibits prolyl hydroxylase activity, and indirectly stabilizes Hif-1α protein [37]. However, overproduction of ROS can lead to oxidative stress, resulting in decreased SOD and T-AOC activity. SOD can also inhibit the activation of NF-κB to limit the inflammatory response [38]. Further, oxidative stress amplifies microglia inflammatory responses, resulting in neuron injury [39][40][41]. In the present study, we found that ROS production was increased in hypoxia-treated microglia (Figures 3(a) and  3(b)), whereas SOD and T-AOC activities were decreased (Figures 3(c) and 3(d)). In contrast, propofol was able to restore antioxidant activity (both SOD and T-AOC) in hypoxia-treated microglia (Figures 3(c) and 3(d)), thus  inhibiting the production of ROS and inflammatory responses. It should be noted that the concentration of propofol was a key factor in this process and that high concentrations (100 μM) were found to reduce antioxidant activity. This might be due to cytotoxicity at high concentrations [42,43]. This decrease in antioxidant activity (both SOD and T-AOC) in hypoxia-treated microglia is consistent with previous studies [9,44].
In addition, overproduction of ROS also results in the loss of mitochondrial membrane potential and aggravates mitochondrial dysfunction [8,45,46]. Furthermore, the effect of propofol on mitochondrial membrane potential and mitochondrial function is related to dose, cell type, and administration route [12,47]. Studies have shown that propofol can prevent the collapse in membrane potential in liver and brain mitochondria during ischemia [11,12]. In this study, we found that propofol could attenuate hypoxiainduced decreases in mitochondrial membrane potential ( Figure 4) but failed to do so at 50 μM concentration.
To summarize, our data show that propofol attenuates hypoxia-induced inflammation in BV2 microglia, in addition to reducing the production of ROS and enhancing antioxidant activity, at least in part by regulating the NF-κB/Hif-1α signaling pathway. Further studies have to be performed to explore the effectiveness of propofol in clinical trials and to standardize the mode of administration with an appropriate dosage.

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

Conflicts of Interest
The authors declare no competing interests.

Authors' Contributions
XP and WY worked on study design, CL and SL handled the study conduct, XP and CL participated in data analysis, XP and SQ wrote the paper, and all authors revised the paper.