Quercus acuta Thunb. Suppresses LPS-Induced Neuroinflammation in BV2 Microglial Cells via Regulating MAPK/NF-κB and Nrf2/HO-1 Pathway

Microglial activation-mediated neuroinflammation is associated with the pathogenesis of neurodegenerative disorders. Therefore, the management of microglial cell activation and their inflammatory response is an important therapeutic approach for preventing neurodegenerative diseases. Quercus acuta Thunb. (QA) (Fagaceae) is a tree found in Korea, China, and Japan. The current study investigated the anti-neuroinflammatory effects of QA and its mechanism of action in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Pretreatment with a methanol extract of dried QA stems (QAE) inhibited the production of nitric oxide and proinflammatory cytokines and decreased the expression of inducible nitric oxide synthase, cyclooxygenase-2 in LPS-stimulated BV2 microglial cells. Furthermore, it inhibited the phosphorylation and degradation of inhibitory κBα and decreased the nuclear translocation and phosphorylation of nuclear factor-κB (NF-κB). Moreover, QAE inhibited the phosphorylation of extracellular signal-regulated kinase, p38 and c-Jun N-terminal kinase, which is known as mitogen-activated protein kinase (MAPK). Additionally, QAE treatment increased heme oxygenase-1 (HO-1) expression by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling, thereby ameliorating LPS-induced intracellular hydrogen peroxide production. Finally, it was found that catechin and taxifolin, two phytochemicals of QAE, also reduced the expression of inflammatory mediators. These findings suggest that QA is beneficial for preventing microglia-mediated neuroinflammatory response through the inhibition of NF-κB, MAPK and the activation of Nrf2/HO-1 signaling pathways.


Introduction
Microglia, innate immune effector cells that reside in the central nerve system (CNS), perform pivotal roles in brain homeostasis. Microglia exert neuroprotective functions in the healthy brain by providing innate immunity, removing apoptotic cells and modifying synaptic connectivity [1]. On the other hand, stimuli such as pathogenic insults, traumatic brain injury and amyloid, tau protein activate the microglia, prompting them to release cytokines, chemokines and reactive oxygen species (ROS) [2]. Microglial activation-driven neuroinflammation eventually causes neuronal cell death and synaptic dysfunction since neurons are vulnerable to these inflammatory mediators [1,3]. Given that increased microglial activation is positively correlated with neuronal dysfunction and abnormal protein aggregation in patients with Alzheimer's and Parkinson's disease, regulating the inflammatory response of microglia is regarded as an important prophylactic target to prevent the development and progress of neurodegenerative disease [4,5].
Microglial cells mediate an inflammatory response against pathogenic stimuli by recognizing pathogen-associated molecular patterns or damage-associated molecular patterns via toll-like receptor (TLR) [6]. Endotoxin binding to TLR on the surface of microglia induces intracellular signaling transduction, which activates the nuclear factor-κB (NF-κB)

Cell Viability Assay
BV2 microglial cells were plated in 96-well plates (30,000 cells/well) to determine cell viability. After treatment, cells were further incubated with CCK-8 (Dojindo Laboratories, Kumamoto, Japan) for 2 h. Absorbance at 450 nm was detected using an automated plate reader (SpectraMax i3, Molecular Devices, Sunnyvale, CA, USA). Relative cell viability was calculated using the following equation: Relative cell viability (%) = Absorbance of the treated group Absorbance of the control group × 100

Measurement of Nitric Oxide (NO) Production
To measure NO production, conditioned media was reacted with the same volume of Griess reagent for 10 min. Absorbance at 550 nm was measured using an automated plate reader (Molecular Devices).

Preparation of Whole Cell Lysates and Nuclear Fraction Extract, and Immunoblot Assay
Cells were lysed with radioimmunoprecipitation (RIPA) buffer containing protease and phosphatase inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany), and incubated on ice for 40 min with vigorous vortex every 10 min. The cell lysate was centrifuged at 15,000× g for 30 min, and the supernatant was collected as whole cell lysates. Nuclear fraction was obtained using a commercial nuclear extraction kit (Thermo Fisher Scientific), according to the manufacturer's instruction. The bicinchoninic acid assay was performed to determine the protein content (Thermo Fisher Scientific). An equal amount of protein was then separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). After blocking with 5% skim milk, the membrane was allowed to reacted with primary antibody overnight at 4 • C, and subsequently incubated with horseradish peroxide conjugated-secondary antibody (Cell Signaling Technology). The chemiluminescence density of the protein of interest was detected by the Alliance Q9 chemiluminescence imager (Uvitec, Cambridge, UK), and densitometric analysis was performed using ImageJ (US National Institutes of Health, Bethesda, MD, USA). Equal protein loading was verified by β-actin (whole cell lysates) or TBP (nuclear fraction) immunoblotting.

RNA Isolation and Real-Time PCR
Total RNA was extracted using QIAzol Lysis Reagent (Qiagen, Hilden, Germany), as directed by the manufacturer. Reverse transcription was conducted using Maxima Reverse Transcriptase (Thermo Fisher Scientific). Real-time PCR was performed using SYBR Green qPCR Master Mix (Thermo Fisher Scientific) and CFX real-time PCR detection system (Bio-Rad, Hercules, CA, USA). Glyceraldehyde-3-phophate dehydrogenase (GAPDH) was used as the reference gene. The primer sequences used for the RT-qPCR reaction are given in Table 1. Relative gene expression was calculated using the 2 −∆∆CT method [20].

Measurement of Cytokine Release
BV2 microglial cells were preincubated with QAE (25-100 µg/mL) for 3 h, and exposed to LPS for 12 h. After treatment, the level of cytokine in cultured media was measured using commercial enzyme-linked immunosorbent assay (ELISA) kit (Thermo Fisher Scientific) according to the manufacturer's instruction.

Immunocytochemistry
Cells were seeded onto coverslips (Marienfeld, Lauda-Königshofen, Germany). After treatment, cells were fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.25% Triton X-100 for 30 min at room temperature. After blocking with 1% bovine serum albumin, cells were incubated with primary antibody (anti-p65) for overnight at 4 • C, and subsequently reacted with Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific) at room temperature for 2 h. After staining nuclei with Hoechst 33342 (Thermo Fisher Scientific), coverslips were mounted with fluorescence mounting solution (Dako, Glostrup, Denmark), and observed under a Lionheart FX microscope (BioTek, Vermont, Winooski, VT, USA).

Measurement of H 2 O 2 Production
After treatment, BV2 microglial cells were stained with H 2 DCF-DA (10 µM) for 30 min. The fluorescence intensity of dichlorofluorescein (DCF) was detected at a wavelength of 485/530 nm (emission/excitation) using an automated plate reader (Molecular Devices).

UHPLC-UV-HRMS Analysis
Phytochemical identification in QAE was performed by a Q-Exactive quadrupole Orbitrap mass spectrometer coupled with Thermo Dionex UltiMate 3000 system (UHPLC-UV-HRMS, Thermo Fisher Scientific). Chromatographic separation was carried out on a Waters Acquity BEH C18 column (2.1 mm i.d. × 100 mm, 1.7 µm) equipped with VanGuard XBridge BEH C18 pre-column (2.1 mm i.d. × 5 mm, 1.7 µm), with the oven temperature maintained at 40 • C. The mobile phase conditions for analysis were composed of 0.1% formic acid (v/v) in water (A) and acetonitrile (B), and the flow rate was maintained at 0.3 mL/min. Full MS and MS/MS spectrums were obtained via UHPLC-UV-HRMS that equipped with a heated electrospray ionization (HESI) interface. The data acquisition and analysis of all data obtained through this study were processed through Xcalibur v.4.2 and Tracefinder v.4.0 software (Thermo Fisher Scientific). In addition, the analysis method used in this study was referred and applied to the previously reported methods [21] and briefly described.

Statistical Analysis
To determine the significance difference among experimental groups, unpaired student's t-test or one-way analysis of variance (ANOVA) was performed. According to results from one-way ANOVA, Tukey's honest difference was conducted as post hoc analysis. All numerical data were expressed as bar chart (mean ± standard deviation) or box plot of at least three independent experiments. p Values under 0.05 were considered as statistically significant. The statistical analysis test was accomplished using GraphPad Prism 6.0 software (GraphPad Software, San Diego, CA, USA).

QAE Inhibited NO Production in LPS-Stimulated BV2 Microglial Cells
First, the effect of QAE on cell viability was assessed to evaluate the cytotoxicity of QAE and the suitable dose on BV2 microglial cells. The final concentration of QAE up to 100 µg/mL (24 h) had no discernible cytotoxic effect on the viability of BV2 microglial cells ( Figure 1a). Furthermore, the NO released in the medium was measured by Griess assay to determine whether QAE possessed any anti-inflammatory activity. Excessive NO production has been linked to the development of neurodegenerative diseases via nitrosative stress, protein misfolding, and neuronal damage [2,8]. After pretreatment with QAE (25-100 µg/mL, 3 h), BV2 microglial cells were exposed to LPS (100 ng/mL) for 24 h. As expected, the LPS treatment significantly increased NO production. Pretreatment with QAE (25-100 µg/mL) reduced NO generation in a dose-dependent manner (Figure 1b). Based on these results, further experiments were carried out at QAE concentrations in the range of 25-100 µg/mL. equipped with a heated electrospray ionization (HESI) interface. The data acquisition and analysis of all data obtained through this study were processed through Xcalibur v.4.2 and Tracefinder v.4.0 software (Thermo Fisher Scientific). In addition, the analysis method used in this study was referred and applied to the previously reported methods [21] and briefly described.

Statistical Analysis
To determine the significance difference among experimental groups, unpaired student's t-test or one-way analysis of variance (ANOVA) was performed. According to results from one-way ANOVA, Tukey's honest difference was conducted as post hoc analysis. All numerical data were expressed as bar chart (mean ± standard deviation) or box plot of at least three independent experiments. p Values under 0.05 were considered as statistically significant. The statistical analysis test was accomplished using GraphPad Prism 6.0 software (GraphPad Software, San Diego, CA, USA).

QAE Inhibited NO Production in LPS-Stimulated BV2 Microglial Cells
First, the effect of QAE on cell viability was assessed to evaluate the cytotoxicity of QAE and the suitable dose on BV2 microglial cells. The final concentration of QAE up to 100 g/mL (24 h) had no discernible cytotoxic effect on the viability of BV2 microglial cells ( Figure 1a). Furthermore, the NO released in the medium was measured by Griess assay to determine whether QAE possessed any anti-inflammatory activity. Excessive NO production has been linked to the development of neurodegenerative diseases via nitrosative stress, protein misfolding, and neuronal damage [2,8]. After pretreatment with QAE (25-100 g/mL, 3 h), BV2 microglial cells were exposed to LPS (100 ng/mL) for 24 h. As expected, the LPS treatment significantly increased NO production. Pretreatment with QAE (25-100 g/mL) reduced NO generation in a dose-dependent manner ( Figure 1b). Based on these results, further experiments were carried out at QAE concentrations in the range of 25-100 g/mL.

QAE Suppressed iNOS and COX-2 Expression in LPS-stimulated BV2 Microglial Cells
iNOS is a key enzyme responsible for inflammatory response by synthesizing NO from L-arginine [22]. The protein expression of iNOS was evaluated by immunoblot assay if the inhibitory effect of QAE on NO production was mediated by iNOS expression. As expected, LPS stimulation (100 ng/mL, 12 h) significantly increased the expression of iNOS (Figure 2a), and pretreatment with QAE (25-100 g/mL) significantly suppressed Figure 1. Methanol extract of Quercus acuta Thunb. stem (QAE) inhibits nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. BV2 microglial cells were pretreated with QAE (25-100 µg/mL) for 3 h, and then exposed to LPS (100 ng/mL) for 24 h. (a) Relative cell viability. After treatment, cell viability was measured using CCK-8 (n = 3). (b) NO production. Produced NO in media was determined by Griess assay (n = 5). Significant versus control, *** p < 0.001; Significant versus LPS alone-treated group, ### p < 0.001; N.S., not significant.

QAE Suppressed iNOS and COX-2 Expression in LPS-Stimulated BV2 Microglial Cells
iNOS is a key enzyme responsible for inflammatory response by synthesizing NO from L-arginine [22]. The protein expression of iNOS was evaluated by immunoblot assay if the inhibitory effect of QAE on NO production was mediated by iNOS expression. As expected, LPS stimulation (100 ng/mL, 12 h) significantly increased the expression of iNOS (Figure 2a), and pretreatment with QAE (25-100 µg/mL) significantly suppressed iNOS protein level. According to protein expression, the mRNA level of iNOS was decreased by QAE pretreatment at 50 and 100 µg/mL (Figure 2b). COX-2 is an enzyme catalyzing the conversion of arachidonic acid to prostaglandins which act as an inflammatory mediator [9]. QAE (100 µg/mL) pretreatment decreased COX-2 protein expression (Figure 2a), and significantly suppressed mRNA expression of COX-2 ( Figure 2b). iNOS protein level. According to protein expression, the mRNA level of iNOS was decreased by QAE pretreatment at 50 and 100 g/mL (Figure 2b). COX-2 is an enzyme catalyzing the conversion of arachidonic acid to prostaglandins which act as an inflammatory mediator [9]. QAE (100 g/mL) pretreatment decreased COX-2 protein expression ( Figure  2a), and significantly suppressed mRNA expression of COX-2 ( Figure 2b).

QAE Inhibited NF-B Signaling Activation in LPS-stimulated BV2 Microglial Cells
To determine the molecular mechanisms of the anti-inflammatory effect of QAE, we investigated the effect of QAE on the NF-B signaling pathway in LPS-stimulated BV2 microglial cells. As expected, LPS treatment (100 ng/mL, 1 h) significantly increased phosphorylation and degradation of IB. However, QAE pretreatment (50 and 100 g/mL, 3 h) significantly inhibited IB phosphorylation and inhibited degradation of IB (Figure 4a). To investigate the effect of QAE on the nuclear translocation of NF-B (p65 subunit), nuclear p65 was detected by immunoblotting and immunofluorescence staining using p65 subunit-specific antibody. LPS stimulation (100 ng/mL, 1 h) enhanced the nuclear translocation of p65, which was considerably inhibited by QAE pretreatment (50 or 100 g/mL, 3 h) (Figure 4b and c). Furthermore, we examined the effect of QAE on phosphorylation at Ser 536 of the p65 subunit by immunoblotting. It is reported that LPS-induced phosphorylation of the p65 subunit at Ser 536 is essential to the elevation of NF-B transcriptional activity [23]. QAE pretreatment (50, 100 g/mL, 3 h) significantly inhibited phosphorylation at Ser 536 of p65 (Figure 4d). Overall, these results suggest that QAE exhibited antiinflammatory activity through NF-B inhibition.

QAE Inhibited NF-κB Signaling Activation in LPS-Stimulated BV2 Microglial Cells
To determine the molecular mechanisms of the anti-inflammatory effect of QAE, we investigated the effect of QAE on the NF-κB signaling pathway in LPS-stimulated BV2 microglial cells. As expected, LPS treatment (100 ng/mL, 1 h) significantly increased phosphorylation and degradation of IκB. However, QAE pretreatment (50 and 100 µg/mL, 3 h) significantly inhibited IκB phosphorylation and inhibited degradation of IκB (Figure 4a). To investigate the effect of QAE on the nuclear translocation of NF-κB (p65 subunit), nuclear p65 was detected by immunoblotting and immunofluorescence staining using p65 subunitspecific antibody. LPS stimulation (100 ng/mL, 1 h) enhanced the nuclear translocation of p65, which was considerably inhibited by QAE pretreatment (50 or 100 µg/mL, 3 h) (Figure 4b,c). Furthermore, we examined the effect of QAE on phosphorylation at Ser 536 of the p65 subunit by immunoblotting. It is reported that LPS-induced phosphorylation of the p65 subunit at Ser 536 is essential to the elevation of NF-κB transcriptional activity [23]. QAE pretreatment (50, 100 µg/mL, 3 h) significantly inhibited phosphorylation at Ser 536 of p65 (Figure 4d). Overall, these results suggest that QAE exhibited anti-inflammatory activity through NF-κB inhibition.

QAE Inhibited Activation of MAPK Signaling Pathway
MAPK signaling is well known to promote the production of inflammatory mediators [24]. To examine whether the anti-inflammatory activity of QAE is mediated by the abrogation of the MAPK signaling pathway, the phosphorylation of ERK, p38, and JNK was measured by immunoblot analysis. LPS stimulation (100 ng/mL, 1 h) significantly increased the phosphorylation of ERK, p38, and JNK. However, the phosphorylation of ERK, p38, and JNK was remarkably reduced in a dose-dependent manner by QAE pretreatment (25-100 µg/mL, 3 h) ( Figure 5). These results suggest that QAE exhibited an inhibitory effect on the inflammatory response via inactivation of MAPK signaling.

UHPLC-UV-HRMS Analysis of Catechin and Taxifolin in QAE
To identify the phytochemicals in QAE, an analysis was performed using UHPLC UV-HRMS. As a result of the analysis, two phenolic components, catechin and taxifoli were identified in QAE. Figure 8 shows the extracted ion chromatograms (EIC), which ar chromatograms for the precursor ion m/z values of each analyte, and MS/MS spectrums The component identified at retention time (tR) of 5.07 min presented precursor ions at m/ 289.0715 [M-H]¯ (Error-0.89 ppm), which was composed of molecular formula C15H14O provided by Orbitrap. In addition, the fragment ions of the MS/MS spectrum were m/ 289.0713, 245.0812, 205.0495, and 125.0228. By comparing these results with previous lit erature, this component was identified as catechin [19,25]. Taxifolin was identified as fo lows. Taxifolin detected at 6.99 min showed a precursor ion at m/z 303.0508 [M-H]¯ (Er ror-0.92 ppm), which consists of the molecular formula C15H12O7 provided by Orbitrap The fragment ions shown in the MS/MS spectrum were m/z 285.0398, 177.0181, an 125.0227. Likewise, it was identified as taxifolin by comparing with the previous literatur [19,25,26]. Both catechin and taxifolin identified in QAE were determined by comparin retention time, measured precursor ion, and MS/MS fragments with the values o Cell viability was determined using CCK-8 (n = 4). (b) NO production. Cells were pretreated with each phytochemical (25-100 µM) for 3 h, and further exposed to LPS (100 ng/mL) for 24 h. Produced NO in media was determined using Griess reagent (n = 4). Relative mRNA expression of (c) iNOS (NOS2), COX-2 (PTGS2), and (d) TNF-α (TNF), IL-1β (IL1B), IL-6 (IL6), and MCP-1 (CCL2). Cells were pretreated with catechin (n = 4) or taxifolin (n = 3), and subsequently stimulated with LPS (100 ng/mL) for 8 h. Significant versus control, *** p < 0.001; Significant versus LPS alone-treated group, # p < 0.05, ## p < 0.01, ### p < 0.001.

UHPLC-UV-HRMS Analysis of Catechin and Taxifolin in QAE
To identify the phytochemicals in QAE, an analysis was performed using UHPLC-UV-HRMS. As a result of the analysis, two phenolic components, catechin and taxifolin were identified in QAE. Figure 8 [19,25]. Taxifolin was identified as follows. Taxifolin detected at 6.99 min showed a precursor ion at m/z 303.0508 [M-H] (Error-0.92 ppm), which consists of the molecular formula C 15 H 12 O 7 provided by Orbitrap. The fragment ions shown in the MS/MS spectrum were m/z 285.0398, 177.0181, and 125.0227. Likewise, it was identified as taxifolin by comparing with the previous literature [19,25,26]. Both catechin and taxifolin identified in QAE were determined by comparing retention time, measured precursor ion, and MS/MS fragments with the values of reference standards, and it was confirmed that the negative ion mode was the optimal condition to for the analysis of both components. reference standards, and it was confirmed that the negative ion mode was the optimal condition to for the analysis of both components.

Discussion
Neuroinflammation has been considered as a significant risk factor for the pathogenesis of neurodegenerative disorders such as Alzheimer's and Parkinson's disease [3,6]. Although the anti-hyperuricemia, antibacterial, antiviral activities of QA have been identified, the beneficial effects of QA on neuroinflammation have not been established [15,[17][18][19]. Therefore, in the present study, we explored the anti-neuroinflammatory properties of QAE using LPS-stimulated BV2 microglial cells.
Prolonged activation of microglia results in the excessive release of cytokines and neurotoxic molecules, which eventually contribute to neurodegeneration [2]. Thus, controlling microglial activation to reduce the emergence of proinflammatory mediators may be advantageous in managing neurodegenerative disease [6]. First, we studied the effect of QAE on the production of proinflammatory mediators in LPS-stimulated BV2 microglial cells. The results revealed that QAE pretreatment significantly inhibited LPS-induced NO production ( Figure 1) and iNOS, COX-2 expressions (Figure 2). Furthermore, it reduced mRNA expression and the release of proinflammatory cytokines including TNF-, IL-1, and IL-6 by LPS stimulation (Figure 3). These results suggested that QAE exhibited an anti-inflammatory effect via regulating the production of proinflammatory mediators.
Previous research identified the five major phytochemicals of QAE (taxifolin, chlorogenic acid, fraxin, catechin, isoquercitrin), and isoquercitrin exhibiting the most excellent antiviral efficacy against HSV-1 viral infection [19]. In other reports, taxifolin and catechin isolated from QA inhibited LPS-induced NO production in RAW 264.7 macrophage cells [18]. The inhibitory effect of phytochemicals on LPS-induced NO production in BV2

Discussion
Neuroinflammation has been considered as a significant risk factor for the pathogenesis of neurodegenerative disorders such as Alzheimer's and Parkinson's disease [3,6]. Although the anti-hyperuricemia, antibacterial, antiviral activities of QA have been identified, the beneficial effects of QA on neuroinflammation have not been established [15,[17][18][19]. Therefore, in the present study, we explored the anti-neuroinflammatory properties of QAE using LPS-stimulated BV2 microglial cells.
Prolonged activation of microglia results in the excessive release of cytokines and neurotoxic molecules, which eventually contribute to neurodegeneration [2]. Thus, controlling microglial activation to reduce the emergence of proinflammatory mediators may be advantageous in managing neurodegenerative disease [6]. First, we studied the effect of QAE on the production of proinflammatory mediators in LPS-stimulated BV2 microglial cells. The results revealed that QAE pretreatment significantly inhibited LPS-induced NO production ( Figure 1) and iNOS, COX-2 expressions (Figure 2). Furthermore, it reduced mRNA expression and the release of proinflammatory cytokines including TNF-α, IL-1β, and IL-6 by LPS stimulation (Figure 3). These results suggested that QAE exhibited an anti-inflammatory effect via regulating the production of proinflammatory mediators.
Previous research identified the five major phytochemicals of QAE (taxifolin, chlorogenic acid, fraxin, catechin, isoquercitrin), and isoquercitrin exhibiting the most excellent antiviral efficacy against HSV-1 viral infection [19]. In other reports, taxifolin and catechin isolated from QA inhibited LPS-induced NO production in RAW 264.7 macrophage cells [18]. The inhibitory effect of phytochemicals on LPS-induced NO production in BV2 microglial cells was tested to see if the five phytochemicals of QAE had an antineuroinflammatory effect. Among the five constituents, catechin and taxifolin showed the most potent anti-inflammatory effect, reducing LPS-induced NO production (Figure 7), and two phenolic compounds in QAE were identified by UHPLC-UV-HRMS analysis (Figure 8).
Furthermore, catechin and taxifolin inhibited LPS-induced mRNA expression of iNOS, COX-2, inflammatory cytokines (TNF-α, IL-1β, IL-6, and MCP-1) (Figure 7b,c). In line with these results, the suppressive effects of catechin and taxifolin on the expression of proinflammatory enzymes in LPS-stimulated BV2 microglial cells were reported previously [27,28]. Based on these findings, the anti-inflammatory activity of QAE is assumed to be connected to the inhibitory efficacy of catechin and taxifolin on the expression of inflammatory mediators.
NF-κB is an essential transcription factor of regulating inflammatory mediators. Signal transduction from TLRs activates IKKα/β, which leads to phosphorylation and degradation of IκB [29]. NF-κB released from IκB translocates to the nucleus and induces the transcription of proinflammatory genes, including iNOS, COX-2, cytokines, and chemokines. In the current study, we revealed that QAE significantly inhibited phosphorylation and degradation of IκB (Figure 4a). Furthermore, immunoblotting results using nuclear protein and immunocytochemistry showed that QAE successfully inhibited the nuclear accumulation of the p65 subunit against LPS stimulation (Figure 4b,c). In addition to nuclear translocation, phosphorylation of p65 on Ser 536 is essential for transcriptional activation of NF-κB. Evidence showed that phosphorylation of p65 on Ser 536 by IKKβ enhances NF-κB transcriptional activity, and p65 phosphorylation (Ser 536 ) is independent of the IκBα regulation [23,30]. In our study, phosphorylation of p65 (Ser 536 ) significantly increased by LPS stimulation, whereas QAE pretreatment effectively reduced the level of p-p65 (Ser 536 ) (Figure 4d). We also investigated the effect of QA on the LPS-induced activation of MAPK signaling. QAE pretreatment significantly inhibited LPS-induced phosphorylation of ERK, p38, and JNK ( Figure 5). MAPK signaling cascades are involved in regulating cell proliferation, cell survival, and inflammation. It has been known that MAPK activates NF-κB signaling through the degradation of IκB [31]. Moreover, studies using chemical inhibitors showed the abrogation of p38, ERK, and JNK signaling suppressed the production of proinflammatory cytokines such as IL-1β, TNF-α in microglia [32,33]. Thus, the inhibition of the MAPK signaling pathway in microglia is beneficial in resolving inflammation in the CNS. These results suggested that QAE inhibited inflammatory gene expression in the microglial cells by blocking MAPK and NF-κB signaling pathways. Intracellular ROS acts as key signaling molecules that are implicated in the inflammatory response of microglia [12]. H 2 O 2 production by LPS-stimulated TLR4 signaling accelerates the activation of NF-κB signaling via phosphorylation and degradation of IκB [11,34]. On the other hand, HO-1, endogenous antioxidant enzyme, and its by-products attenuate TLR4 signaling transduction by inhibiting ROS generation in macrophages [12]. Accordingly, small molecules that activate the Nrf2/HO-1 pathway simultaneously regulate the inflammatory response in BV2 microglial cells [13]. In our study, QAE treatment significantly increased mRNA and the protein expression of HO-1 in a dose-dependent manner, which ameliorated LPS-induced cellular H 2 O 2 generation ( Figure 6).
Nrf2 is a major transcriptional factor regulating the expression of antioxidant genes such as HO-1, GCLC, and SESN2. Nrf2 phosphorylation at Ser 40 results in translocation to the nucleus. Finally, Nrf2 binds to the antioxidant response element (ARE), and transactivates antioxidant genes, thereby serving cellular redox homeostasis [35]. Apart from the antioxidant response, the anti-inflammatory properties of Nrf2 via the inhibition of the NF-κB pathway and blocking transcription of inflammatory mediators have also been suggested [36]. Representatively, chromatin immunoprecipitation-seq (ChIP-seq) study in macrophages revealed that Nrf2 inhibited the transcription of proinflammatory cytokines by binding to its promoter regions [37]. In our results, QAE successfully activated the Nrf2 signaling pathway by inducing its phosphorylation and nuclear translocation ( Figure 6). Taken together, our results suggested that the effect of QAE on Nrf2/HO-1 activation contributes to QA's anti-inflammatory efficacy.
Overall, our results showed the anti-neuroinflammatory effect of QAE in LPS-stimulated BV2 microglial cells by reducing the expression of proinflammatory enzymes and mediators. These activities were related to NF-κB, MAPK inhibition, and Nrf2/HO-1 activation.
Although the present study revealed cellular efficacy and molecular mechanisms of QA on the microglial inflammatory response, efficacy evaluation using neurodegenerative disease experimental animal models needs to be further established for clinical application.

Conclusions
In the present study, we demonstrated that QAE exhibited anti-inflammatory effects on LPS-stimulated BV2 microglial cells by reducing the expression of proinflammatory enzymes and mediators including iNOS, COX-2, NO, TNF-α, IL-1β, and IL-6. Moreover, we revealed that these inhibitory effects were mediated by inhibiting NF-κB and MAPK signaling pathways. Additionally, we found antioxidative stress efficacy of QAE against LPS-stimulation via the activation of the Nrf2/HO-1 pathway. Thus, QA may be a promising candidate for managing neuroinflammation-mediated neurodegenerative diseases.