Processed Scutellaria baicalensis Georgi Extract Alleviates LPS-Induced Inﬂammatory and Oxidative Stress through a Crosstalk between NF- κ B and KEAP1/NRF2 Signaling in Macrophage Cells

: Prolonged inﬂammation results in chronic diseases that can be associated with a range of factors. Medicinal plants and herbs provide synergistic beneﬁts based on the interaction of multiple phytochemicals. The dried root of Scutellaria baicalensis Georgi and its compounds possess anti-inﬂammatory, anti-oxidative, and anticancer effects. Processing is a traditional method to achieve clinical beneﬁts by improving therapeutic efﬁcacy and lowering toxicity. In this study, we investigated the anti-inﬂammatory and anti-oxidant effect of processed Scutellaria baicalensis Georgi extract (PSGE) against lipopolysaccharide (LPS) stimulated RAW 264.7 cells. Data using Griess assay and ELISA showed that PSGE decreased nitric oxide and prostaglandin E2 (PGE 2 ) levels against LPS. PSGE treatment up-regulated 15-hydroxyprostaglandin dehydrogenase (PGDH), while cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase (mPGES)-1 expression did not change. Interestingly, PGE 2 inhibition was regulated by prostaglandin catabolic enzyme 15-PGDH rather than COX-2/mPGES-1, enzymes essential for PGE 2 synthesis. Additionally, PSGE-suppressed LPS-induced IL-6 and TNF- α production through NF- κ B signaling. NF- κ B release from an inactive complex was inhibited by HO-1 which blocked I κ B α phosphorylation. The ROS levels lowered by PSGE were measured with the H 2 DCFDA probe. PSGE activated NRF2 signaling and increased antioxidant Hmox1 , Nqo1 , and Txn1 gene expression, while reducing KEAP1 expression. In addition, pharmacological inhibition of HO-1 conﬁrmed that the antioxidant enzyme induction by PSGE was responsible for ROS reduction. In conclusion, PSGE demonstrated anti-inﬂammatory and anti-oxidant effects due to NRF2/HO-1-mediated NF- κ B and ROS inhibition.


Introduction
Inflammation is the body's nonspecific response to infectious or non-infectious etiologies, which accompanies pain, heat, redness and swelling [1,2]. The triggered acute inflammatory responses are resolved by the innate immune cells, such as macrophages and neutrophils [3]. However, prolonged inflammation, also referred to as chronic inflammation, causes additional damage to organs including the brain, lungs, heart, digestive tract, kidneys, intestinal tract, and reproductive system. Failure to prevent chronic inflammation results in chronic diseases such as type 2 diabetes, cancer, rheumatoid arthritis, neurodegenerative, cardiovascular and bowel diseases [3]. Dysregulation of nuclear factor kappa-B

Cell Viability Assay
Cell cytotoxicity after PSGE treatment was examined by the MTT assay. A density of 1.4 × 10 4 cells/well were seeded and incubated for 24 h. Cells were treated with several concentrations of PSGE for 24 h. Media was removed and media containing MTT solution (0.5 mg/mL) was added per well, and the cells were incubated for another 4 h. Next, the MTT reagent and media were discarded and 100 µL DMSO was added to dissolve the formed formazan crystals. Finally, the plate was gently shaken on a shaker, and absorbance was measured at 570 nm with a microplate reader (Biotek, VT, USA).

Nitric Oxide (NO) Assay
The amount of NO was determined by the levels of nitrite with the cultured RAW 264.7 cell supernatant using Griess reagent containing 0.1% N-(1-Naphthyl) ethylenediamine dihydrochloride in double-distilled water and 1% sulfanilamide in 5% phosphoric acid. Briefly, RAW 264.7 cells (5 × 10 5 cells/well) were seeded and incubated for 24 h. Following pre-treatment with different concentrations of PSGE and 20 µM DXM for 2 h, the cells were stimulated with LPS (1 µg/mL) for an additional 24 h. Culture media was collected, and the amount of nitrite was measured using Griess reagent. Griess reagent was added to an equal volume and the plate was gently agitated for 10 min on a shaker at room temperature (RT). 540 nm absorbance was monitored by a microplate reader and the concentration of nitrite was determined from a standard curve generated from known sodium nitrite concentrations.

IL-6, TNF-α and PGE 2 Assay
RAW 264.7 cells (1 × 10 5 cells/well) were pretreated with different concentrations of PSGE and DXM 20 µM for 2 h and then exposed to LPS 1µg/mL for 24 h. The supernatant of each well was collected and centrifuged at 1500 rpm at 4 • C for 10 min. IL-6 and TNF-α concentrations were measured using mouse IL-6 and TNF-α ELISA kit (Invitrogen, Carlsbad, CA, USA) and PGE 2 using mouse PGE 2 ELISA kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer's instructions.

Western Blotting
Cells were rinsed with chilled PBS and lysed in RIPA containing 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, 150 mM NaCl, 50 mM Tris HCL (pH 7.4), 2 mM EDTA and I X protease inhibitor cocktail. The samples were incubated on ice for 10 min and vortexed for 10 s after an interval time for lysis. The cell debris was cleared by centrifugation at 14,000× g for 15 min. Nuclear and cytoplasmic proteins were separated by NE-PER nuclear and cytoplasmic extraction kit (Thermo Fisher Scientific, Rockford, IL, USA) following the manufacturer's instructions. The protein concentration was determined by Coomassie dye-binding (Bradford) assay using the colorimetric protein assay kit (Biorad, Hercules, CA, USA). Cell proteins were separated by SDS-PAGE and transblotted to nitrocellulose membranes. The membranes were then blocked for 1 h at RT in 5% skim milk, followed by an overnight incubation at 4 • C with a specific primary antibody. The next day, the membranes were washed and incubated for an additional 1 h with HRP-conjugated secondary antibody at RT after washing three times with TBST. Bands were detected by ECL (LPS Solution, Daejeon, Korea), and band intensities were analyzed with Image J.

Cellular Reactive Oxygen Species (ROS) Assay
The Cellular ROS Assay Kit (Abcam, Cambridge, UK) is a DCFDA/H 2 DCFDA dyebased assay to detect intracellular hydroxyl radical, peroxy radical, and other ROS activity.

Statistical Analysis
Data is shown as means ± SEM and are pooled from three independent experiments. Statistical analysis was performed by ANOVA with Tukey's Multiple Comparison Test using software GraphPad Prism 5.1. (GraphPad Software, San Diego, CA, USA). p-values < 0.05 were considered statistically significant.

LC-MS Analysis
LC-MS was used to analyze the retention time and mass pattern of SGE or PSGE compared with standards. The standards baicalin, baicalein, and wogonin were detected at 7.56 min, 12.74 min, and 16.38 min respectively ( Figure 1A-E). According to the LC-MS quantitative analysis results, the contents of baicalin, baicalein, and wogonin were 219.89 ± 1.24 mg/g, 10.21 ± 0.14 mg/g, and 2.55 ± 0.05 mg/g in SGE, and 212.79 ± 0.12 mg/g, 15.41 ± 0.12 mg/g, and 4.59 ± 0.04 mg/g in PSGE respectively.

PSGE Modulated the Release of NO and PGE 2
In order to examine the cytotoxicity of PSGE, we performed a MTT assay in RAW 264.7 cells. Evaluation on cells treated with different doses (50 to 1000 µg/mL) of PSGE were evaluated. PSGE did not cause cell toxicity up to 400 µg/mL ( Figure 2A). Experiments with PSGE concentration ranging from 50 to 200 µg/mL and DXM 20 µM, as positive control, were performed in the study.
We investigated whether PSGE regulated the formation of inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE 2 ) in LPS-challenged RAW 264.7 cells. PSGE treatment considerably inhibited NO and PGE 2 production, in a dose dependent manner, compared to LPS-treated cells ( Figure 2B,C). PSGE (200 µg/mL) suppressed NO production by 0.28-fold and almost completely diminished PGE 2 production when compared with LPS-treated cells. Moreover, it is notable that PSGE caused more extensive inhibition compared to the positive control DXM. We next examined the effect of PSGE on the inducible nitric oxide synthase (iNOS) and the cyclooxygenase (COX)-2 expression levels. The expression of iNOS was significantly increased by LPS but decreased with PSGE treatment dose-dependently ( Figure 2D,E). Despite the significant PGE 2 inhibition, COX-2 levels were unaffected suggesting that PSGE might target the downstream of COX-2 in the PGE 2 synthesis cascade ( Figure 2D,F). The results of microsomal prostaglandin E synthase (mPGES)-1 expression, a terminal synthase of PGE 2 , showed that mPGES-1 also did not influence PGE 2 inhibition by PSGE ( Figure 2D,G). We next examined the expression levels of 15-hydroxyprostaglandin dehydrogenase (PGDH), a PGE 2 degrading enzyme. LPS-treated cells showed 0.5-fold decrease in 15-PGDH levels compared to control. PSGE

PSGE Modulated the Release of NO and PGE2
In order to examine the cytotoxicity of PSGE, we performed a MTT assay in RAW 264.7 cells. Evaluation on cells treated with different doses (50 to 1000 µg/mL) of PSGE were evaluated. PSGE did not cause cell toxicity up to 400 µg/mL (Figure 2A). Experiments with PSGE concentration ranging from 50 to 200 µg/mL and DXM 20 µM, as positive control, were performed in the study.

PSGE Modulated the Release of NO and PGE2
In order to examine the cytotoxicity of PSGE, we performed a MTT assay in RAW 264.7 cells. Evaluation on cells treated with different doses (50 to 1000 µg/mL) of PSGE were evaluated. PSGE did not cause cell toxicity up to 400 µg/mL (Figure 2A). Experiments with PSGE concentration ranging from 50 to 200 µg/mL and DXM 20 µM, as positive control, were performed in the study.

PSGE Regulated the NF-κB-Mediated Inflammatory Responses
The effect of PSGE on pro-inflammatory cytokines such as TNF-α and interleukin 6 (IL-6) was investigated in LPS stimulated RAW 264.7 cells. PSGE significantly suppressed IL-6 levels at 100 and 200 µg/mL and TNF-α levels at 200 µg/mL ( Figure 3A,B). Notably, at 200 µg/mL, PSGE inhibited IL-6 more strongly (0.55-fold), similar to DXM, compared to TNF-α (0.75-fold). Based on these anti-inflammatory effects of PSGE, we determined whether PSGE could modulate nuclear translocation of NF-κB, a transcription factor associated with pro-inflammatory responses. PSGE significantly down-regulated the nuclear expression of NF-κB p65 by 0.70-fold (at 100 µg/mL) and 0.57-fold (at 200 µg/mL) respectively compared to LPS-treated cells ( Figure 3C,D). Inhibitor kappa B-alpha (IκBα) phosphorylation involved in the regulatory steps for LPS-mediated NF-κB activation was determined by Western blot analysis. IκBα phosphorylation increased by 40-fold in LPStreated cells but PSGE treatment significantly attenuated this effect dose-dependently ( Figure 3E,F). Next, we examined the role of Heme Oxygenase-1 (HO-1) on NF-κB p65 nuclear translocation. PSGE treatment augmented HO-1 expression by 1.55-fold (at 100 µg/mL) and 3-fold (at 200 µg/mL) compared to LPS-treated cells ( Figure 3E,G). Our data suggested that PSGE upregulated HO-1 expression, maintained the retention of IκBα/NF-κB complex, and prevented the release of NF-κB into the nucleus.  Production of (A) IL-6 and (B) TNF-α levels were measured by ELISA. RAW 264.7 cells were pre-treated with indicated concentration of PSGE or DXM for 2 h followed by LPS for 24 h. (C) Western blot analysis of nuclear and cytosolic fractions using NF-κB p65 antibody, (D) quantitative analysis of nuclear NF-κB p65 relative to Lamin A/C, (E) Protein expression and quantitative analysis of (F) p-IκBα relative to IκBα and (G) HO-1 relative to β-actin. RAW 264.7 cells were pretreated with the indicated concentrations of PSGE and DXM for 2 h followed by LPS for 2 h. Lamin A/C and HSP90 were used as cytosolic and nuclear markers. DXM as positive control. Data shown represent mean ± SEM (n = 3). (### p < 0.001 vs. control; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. LPS).

PSGE Activated the NRF2-Mediated Antioxidant Enzymes
PSGE significantly protected RAW 264.7 cells against intracellular accumulation of reactive oxygen species (ROS) caused by LPS. Pretreatment with 100 and 200 µg/mL PSGE before 24 h incubation with LPS, inhibited ROS generation by 0.64 and 0.44-fold ( Figure 4A). The interaction between nuclear factor E2-related factor 2 (NRF2) and Kelch-like ECHassociated protein 1 (KEAP1) regulates antioxidant gene expression [23]. PSGE 200 µg/mL treatment decreased KEAP1 expression significantly by 0.46-fold compared to only LPStreated cells (Figure 4B,C). Consequently, we examined whether PSGE could induce NRF2 nuclear translocation when treated with LPS. PSGE exhibited a 1.9-fold (at 100 µg/mL) and 4-fold (at 200 µg/mL) increase in nuclear NRF2 levels ( Figure 4D,F). However, KEAP1 and nuclear NRF2 expression levels did not show any significant change with LPS stimulation (Figure 4B-F). Further results showed NRF2-dependent induction of antioxidant Txn1, Hmox1, and Nqo1 genes with PSGE treatment. PSGE ameliorated the significantly decreased Txn1 (0.53-fold), Hmox1 (0.71-fold), and Nqo1 (0.58-fold) gene expression caused by LPS. At 200 µg/mL, PSGE upregulated Txn1, Hmox1, and Nqo1 mRNA levels by 3.6, 3.0, and 2.1fold ( Figure 4G-I). In order to determine whether PSGE modulated antioxidant enzymes lead to reduction of ROS levels, RAW 264.7 cells were pretreated with SnPPIX (a HO-1 inhibitor) followed by PSGE and LPS. The SnPPIX reversed the inhibitory effects of PSGE on ROS generation after LPS stimulation ( Figure 4J). These data suggest that PSGE elevated antioxidant enzymes to attenuate LPS-induced ROS production via NRF2-KEAP1 pathway. µg/mL treatment decreased KEAP1 expression significantly by 0.46-fold compared to only LPS-treated cells ( Figure 4B,C). Consequently, we examined whether PSGE could induce NRF2 nuclear translocation when treated with LPS. PSGE exhibited a 1.9-fold (at 100 µg/mL) and 4-fold (at 200 µg/mL) increase in nuclear NRF2 levels ( Figure 4D,F). However, KEAP1 and nuclear NRF2 expression levels did not show any significant change with LPS stimulation (Figure 4B-F). Further results showed NRF2-dependent induction of antioxidant Txn1, Hmox1, and Nqo1 genes with PSGE treatment. PSGE ameliorated the significantly decreased Txn1 (0.53-fold), Hmox1 (0.71-fold), and Nqo1 (0.58-fold) gene expression caused by LPS. At 200 µg/mL, PSGE upregulated Txn1, Hmox1, and Nqo1 mRNA levels by 3.6, 3.0, and 2.1-fold ( Figure 4G-I). In order to determine whether PSGE modulated antioxidant enzymes lead to reduction of ROS levels, RAW 264.7 cells were pretreated with SnPPIX (a HO-1 inhibitor) followed by PSGE and LPS. The SnPPIX reversed the inhibitory effects of PSGE on ROS generation after LPS stimulation ( Figure 4J). These data suggest that PSGE elevated antioxidant enzymes to attenuate LPS-induced ROS production via NRF2-KEAP1 pathway.  RAW 264.7 cells were pre-treated with indicated concentration of PSGE or DXM for 2 h followed by LPS for 24 h.(B) Protein expression and quantitative analysis of (C) KEAP1 relative to β-actin, (D) Western blot analysis of nuclear and cytosolic fractions using NRF2 antibody, quantitative analysis of (E) cytosolic NRF2 relative to β-actin, (F) nuclear NRF2 relative to Lamin A/C. RAW 264.7 cells were pre-treated with indicated concentration of the PSGE or DXM for 2 h followed by LPS for 30 min. The levels of (G) Hmox1, (H) Nqo1 and (I) Txn1 mRNA expression were analyzed by RT-qPCR. RAW 264.7 cells were pre-treated with indicated concentration of the PSGE or DXM for 2 h followed by LPS for 2 h. (J) Changes in ROS levels by SnPPIX. RAW 264.7 cells were pre-treated with indicated concentration of SnPPIX (HO-1 inhibitor) for 1 h and then DXM or PSGE for 2 h followed by LPS for 24 h. Lamin A/C and HSP90 were used as cytosolic and nuclear markers. DXM as positive control. Data shown represent mean ± SEM (n = 3). (## p < 0.01 and ### p < 0.001 vs. control; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. LPS).

Discussion
The pathogenesis of chronic inflammatory diseases such as type 2 diabetes, cancer, rheumatoid arthritis, neurological, and cardiovascular and inflammatory bowel diseases are linked with inflammation and oxidative stress [24]. Interaction of multiple phytochemicals in medicinal plants and herbs provide synergistic effects on anti-inflammatory and antioxidant activity [25]. A large number of studies have determined the pharmacological activities of SGE and its chemical constituents, such as anti-viral, anti-tumor, anti-bacterial, antioxidant, anti-inflammatory, hepatoprotective, and neuroprotective activities [26]. Processing is a traditional herbal method to achieve clinical benefits by improving therapeutic efficacy and also by lowering toxicity [27]. The alteration in therapeutic and toxic effects induced by processing is suggested to be related with structural transformation such as hydrolysis, oxidation, decomposition, isomerization, and other chemical reactions [28]. Comparative analysis of the major flavonoids, baicalin, baicalein, and wogonin contained in SGE and PSGE suggested that baicalin content was higher in SGE than PSGE. Interestingly, baicalein and wogonin contents were higher in PSGE than SGE. A report on the change in chemical components of SG after processing showed that stir-frying with alcohol and stir-frying till charred decreased baicalin but increased baicalein and wogonin contents [29]. Earlier studies suggested that the high temperature inhibited glucuronidase activity and prevented hydrolysis of baicalin or wogonin in SG [30,31]. However, understanding the diverse factors influencing glucuronidase activity remains unclear, since it has been reported that baicalin, baicalein, and wogonin contents are increased by heat processing [21,32].
NO is considered as an inflammatory mediator and regulator. In response to inflammatory stimuli, iNOS synthesize NO, inducing proinflammatory and destructive effects that lead to pathological conditions [33]. PSGE pretreatment suppressed NO increase after LPS treatment, which was considerably more potent than DXM treatment ( Figure 2B). PGE 2 is recognized as a bioactive lipid that mediates proinflammatory and immunomodulatory effects. In response to inflammatory stimuli, COX-2 and mPGES-1 mediate concomitant generation of PGE 2 . The elevated PGE 2 levels can also be inactivated by the catabolic enzyme, 15-PGDH [34]. Our data showed that PSGE strongly suppressed PGE 2 production to a greater extent than DXM, while COX-2 and mPGES-1 expression remained unchanged ( Figure 2C,D,F,G). Interestingly, PSGE promoted 15-PGDH expression, which in turn impaired PGE 2 synthesis ( Figure 2D,H). However, previous studies reported that the regulatory effects of SG and PSG on PGE 2 production are dependent on COX-2 downregulation, which remains controversial [35,36]. Other studies have shown that thiazolidinediones pioglitazone and rosiglitazone inhibited PGE 2 production in A427 and A549 lung cancer cells by the catabolic 15-PGDH rather than the synthetic COX-2/mPGES-1 [37]. Further study is needed to fully understand how 15-PGDH expression, altered by PSGE, leads to PGE 2 inhibition without significant change in COX-2 expression. Additionally, the ability of PSGE to increase 15-PGDH may offer therapeutic benefits without resulting in cardiotoxicity known in selective COX-2 inhibitors. PSGE also demonstrated anti-inflammatory effects by decreasing IL-6 and TNF-α release ( Figure 3A,B).
NF-κB is a transcription factor that plays a central role in regulating a large array of genes during immune response and inflammation [38]. NF-κB activity is tightly regulated by IκBα binding in the cytoplasm. Inflammatory stimuli induce IκBα phosphorylation and degradation result in NF-κB nuclear translocation [39]. Our data demonstrated that PSGE inhibition on IL-6 and TNF-α levels involved the prevention of NF-κB release from an inactive complex due to dephosphorylation of IκBα ( Figure 3A-F). Studies have suggested that HO-1 acts on IκB phosphorylation and inhibits activation of NF-κB dependent gene expression [40][41][42]. PSGE treatment increased HO-1 expression, which correlated with decreased IκBα phosphorylation ( Figure 3E,F). These data support the idea that the anti-inflammatory effect of PSGE is mediated by NF-κB regulation via HO-1. The NF-κB and NRF2 pathways play an integral role to maintain redox balance and inflammatory homeostasis upon disruption by cellular stressors. The NRF2 activation is negatively regulated by KEAP1, which promotes ubiquitination and degradation of NRF2. Under stress, KEAP1 is modified and allows NRF2 to translocate to the nucleus where the transcription levels of antioxidant enzymes such as HO-1, NQO1, and TRX1 are activated. The KEAP1/NRF2 signaling pathway negatively regulates the NF-κB-induced cytokine expression by inhibiting intracellular ROS levels [23,43]. Many recent studies provide compelling evidence for the antioxidant and anti-inflammatory role of HO-1 [44]. Our data showed that PSGE pretreatment prevented LPS stimulated increase in ROS production. This was achieved through NRF2 dissociation from KEAP1, leading to nuclear translocation and accumulation of NRF2 ( Figure 4A-F). Furthermore, considerable enrichment of nuclear NFR2 facilitated antioxidant enzyme upregulation and provided robust protection against oxidative stress. We used SnPPIX to confirm whether the antioxidant effect of PSGE against LPS triggered ROS generation was obtained by enhancement of HO-1. HO-1 inhibition restored intracellular ROS levels suppressed by PSGE ( Figure 4G-J). Previous studies on LPS-induced experimental models have shown that SG components such as baicalein, apigetrin, and baicalin alleviate elevated inflammatory cytokines by NF-κB regulation and oxidative damage by HO-1 induction [45][46][47]. Here, we identified that HO-1 may play dual roles in anti-inflammatory and anti-oxidative effects of PSGE mediated by crosstalk between NF-κB and the KEAP1/NRF2 signaling pathway.

Conclusions
PSGE shows synergistic effect by targeting both inflammatory and oxidative stress conditions. The pharmacological functions of PSGE were due to inhibiting NF-κB activation and increasing nuclear NRF2 accumulation. NRF2 activated by PSGE mediated HO-1 upregulation, which resulted in preventing IκBα phosphorylation and ROS production. In addition, PGE 2 production by PSGE involved catabolic 15-PGDH expression rather than synthetic COX-2/mPGES-1 expression. The dual function of PSGE may serve as a potential drug target for chronic inflammatory diseases.