Anti-Inflammatory Effects of Heat-Processed Artemisia capillaris Thunberg by Regulating IκBα/NF-κB Complex and 15-PGDH in Mouse Macrophage Cells

Growing evidence suggests that dietary nutrients in herbs and plants are beneficial in improving inflammatory disorders. Artemisia capillaris Thunberg (AC) is a traditional herbal medicine widely used in East Asia to treat pain, hepatotoxicity, and inflammatory disorders. Heat processing is a unique pharmaceutical method used in traditional herbal medicine to enhance the pharmacological effects and safety of medicinal plants. This study demonstrates the anti-inflammatory effects of heat-processed AC (HPAC) in lipopolysaccharide- (LPS-) treated mouse macrophage cells. HPAC reduced LPS-induced inflammatory mediators such as IL-6, IL-1β, TNF-α, NO, and PGE2 in RAW 264.7 cells. Interestingly, 15-PGDH appears to play a pivotal role rather than COX-2 and mPGES-1 when HPAC regulated PGE2 levels. Meanwhile, HPAC showed anti-inflammatory effects by blocking IκBα phosphorylation and NF-κB nuclear translocalization. Also, we found that HO-1 upregulation was mediated by the mitogen-activated protein kinase (MAPK) pathways in HPAC-treated RAW 264.7 cells. And, in RAW 264.7 cells challenged with LPS, HPAC restored HO-1 expression, leading to NF-κB inhibition. Through further experiments using specific MAPK inhibitors, we found that, in response to LPS, the phosphorylated IκBα and activated NF-κB were attenuated by p38 MAPK/HO-1 pathway. Therefore, HPAC targeting both the IκBα/NF-κB complex and 15-PGDH may be considered as a potential novel anti-inflammatory agent derived from a natural source.


Introduction
Inflammation is a defensive mechanism which acts by removing harmful stimuli such as pathogens, damaged cells, and toxic compounds. It consists of several processes mediated by activated inflammatory and immune cells, including macrophages and monocytes, and incorporates a complex series of reactions regulated by cytokines, growth factors, nitric oxide (NO), and prostaglandins (PGs) produced by active macrophages [1,2]. However, uncontrolled acute inflammation may become chronic, contributing to numerous chronic inflammatory diseases, including arthritis, autoimmune diseases, atherosclerosis, and chronic hepatitis [3,4]. ere is growing evidence that plant foods rich in dietary nutrients are beneficial by inhibiting inflammatory mediators and treating diseases related to such factors [5,6]. Omega-3 fatty acid which is found in several types of nuts and seeds has been reported to protect against autoimmune diseases and atherosclerosis [7]. Curcumin found in Curcuma longa has shown potential effects on diseases such as rheumatoid arthritis, inflammatory bowel disease, and pancreatitis [8]. Ginger contains many phenolic compounds, such as gingerols and shogaols, which have been effective in reducing pain in patients with knee osteoarthritis [9]. Studies have described plant-derived dietary flavonoids, such as quercetin and kaempferol, found in different fruits and vegetables exert anti-inflammatory effects by regulating signaling pathways associated with inflammation [10,11].
Previous studies have shown that extracts of AC, Artemisia apiacea Hance, and Artemisia iwayomogi inhibit lipopolysaccharide-(LPS-) induced inflammatory factors by suppressing NF-κB activation in human HepG2 cells and mouse RAW264.7 cells [31][32][33][34]. However, reports on the effect or mechanism of heat-processed Artemisia capillaris unberg (HPAC) are scarce. e purpose of this study is to evaluate, for the first time, the anti-inflammatory effect of HPAC in LPS-treated mouse macrophage cells and determine the underlying molecular mechanism.

Preparation of Heat-Processed Artemisia capillaris
unberg Extract. Dried AC was purchased from Sunilcrudedrugs (Hongcheon, Korea). e procedure to prepare HPAC follows a previous report with modification [35]. AC was soaked in 30% EtOH for 30 min. AC was roasted in a convection oven (JSOF-150, JS Research Inc., Korea) for 1 h 20 min in 200°C. To prevent the herb from burning, the herb was turned over every 5 min while roasting. HPAC with 2 L of 30% ETOH was boiled for 2 h in 100°C, following filtration and evaporation. e lyophilized HPAC was successively extracted with a yield (w/w) of 8.22%. e voucher specimen (HPAC: BON190527.145) was stored at the herbarium of Korean Medicine at Semyung University. e lyophilized powders were solubilized and diluted with DMSO before treatment.
e constituents of the HPAC extract were evaluated using GC-MS analysis. GC-MS analysis was performed on an Agilent 6890N GC system interfaced with a Leco Pegasus IV Time of flight Mass Spectrometer. e electron energy was −70 eV, and the ion source temperature was 220°C. Each sample (1 μL, dissolved in MeOH) was injected in split mode (10 : 1) at 280°C and separated through a capillary column of DB-5MS (30 × 0.25 × 0.25) (Agilent J&W column). e initial oven temperature was 30°C, which was increased to 300°C at 10°C/min. Carrier gas (Helium) flow was 0.8 mL/min.

Cell
Culture. RAW 264.7 cells were obtained from Korean Cell Line Bank (KCLB) and incubated in highglucose DMEM supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 μg/mL streptomycin under an atmosphere containing 5% CO 2 at 37°C in an incubator.

MTT Cytotoxicity Assay.
e cytotoxicity of HPAC on RAW 264.7 cells was assessed via colorimetric MTT assay. RAW 264.7 cells were grown at a density of 1 × 10 4 cells per well in 96-well plate. After 24 h, cells were then treated with different concentrations of HPAC and incubate for 24 hr. Following treatment, 20 μL of MTT (5 mg/mL) solution was added to each well and further incubated for 4 h at 37°C. Next, the media (containing MTT solution) were removed from each well and 100 μL DMSO was added to each well to dissolve the resulting formazan crystals. Finally, the plate was smoothly agitated for 10 min on a shaker, and the absorbance was then measured at 570 nm using a microplate reader (BioTek, Winooski, VT, USA). e results were shown as relative cell viability referred to as control (equal to 100%). e supernatants were then centrifuged at 12000 rpm at 4°C for 5 min to discard cell debris and the remnant media was collected. e levels of IL-6, TNF-α, and IL-1β were measured by enzyme-linked immunosorbent assay using ELISA kit (Invitrogen, Carlsbad, CA, USA) and PGE 2 were analyzed using ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Extraction of Nuclear and Cytosolic Fraction.
Cytoplasmic and nuclear extracts were prepared using a Nuclear and Cytoplasmic Extraction Reagents kit ( ermo Fisher Scientific, Rockford, IL, USA) following the manufacturer's instructions. Briefly, the RAW 264.7 cells were seeded in 6-well plates at a density of 5 × 10 5 cells/well and incubated for 24 h. Next, the cells were pretreated with different concentrations of HPAC for 2 h and then stimulated with LPS for another 2 h. e cells were then harvested with PBS and centrifuged. Next, an ice-cold cytoplasmic protein extraction solution was added and centrifuged to separate the cytoplasmic extract. en, the cytoplasmic extract was transferred to clean prechilled tubes and the pellets produced were prepared for nuclear extraction by adding ice-cold nuclear protein extraction solution.

Western
Blotting. Cells were lysed in RIPA lysis buffer (25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)) with protease inhibitor cocktail (Roche, Mannheim, Germany). e protein concentration was determined using the bicinchoninic acid protein assay kit ( ermo Scientific, Rockford, IL, USA). Total protein lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. e membranes were then blocked for 1 h at room temperature in 5% skim milk, followed by an overnight incubation at 4°C with a specific primary antibody. e next day, the membranes were washed and incubated for an additional 1 h with HRP-conjugated secondary antibody (1 : 5000) at room temperature after thoroughly washing three times with TBST. Bands were detected by ECL (LPS Solution, Daejeon, Korea), and band intensities were quantified using ImageJ gel analysis software.

Statistical Analysis.
Statistical calculations were done in GraphPad Prism version 5. Results are presented as means ± SEM. Data were analyzed by one-way ANOVA followed by post hoc Tukey's test. p < 0.05 indicates statistical significance.

Cell Viability and Cytokine
Production. RAW 264.7 cells were treated with different concentrations of HPAC (5 μg/mL to 500 μg/mL) and cell viability was performed by MTT assay. After HPAC treatment, significant cell toxicity was observed at 400 μg/mL, and lower concentrations (5 μg/mL to 200 μg/mL) did not show significant changes (Figure 2(a)). erefore, the concentrations of HPAC ranging from 50 μg/mL to 200 μg/mL were employed for further study. Dexamethasone (DXM), a classic steroidal anti-inflammatory and immunomodulatory drug, was included as the positive control in the study [36]. TNF-α, IL-6, and IL-β cytokines play essential roles in inflammatory responses; thus, we investigated whether HPAC could inhibit secretion of these cytokines in macrophages stimulated by LPS. After LPS activation, the results show that IL-6 (147-fold), IL-β (68-fold), and TNF-a (93-fold) levels significantly increased compared to control (Figures 2(b)-2(d)). HPAC treatment suppressed the production of these cytokines in a dose-dependent manner; however, significance was achieved only at 200 ug/mL (Figures 2(b)-2(d)). e levels of IL-6 (0.4-fold), IL-β (0.5-fold), and TNF-a (0.8-fold) changed significantly in the HPAC-treated group (200 ug/mL) compared to the LPS-treated group. After LPS stimulation, the HPAC-treated group demonstrated a lesser effect on inhibiting TNF-α and IL-β production compared to the DXM-treated group (Figures 2(c) and 2(d)). However, HPAC treatment showed stronger IL-6 inhibition in comparison to DXM at 200 μg/mL (Figure 2(b)). ese results indicate that HPAC inhibits the LPS-induced release of proinflammatory cytokines in RAW 264.7 cells.

HPAC Regulates NF-κB Nuclear Translocation.
NF-κB has been reported to be a positive regulator of numerous inflammatory mediators and proinflammatory Evidence-Based Complementary and Alternative Medicine cytokines [37,38]. So we hypothesized that the inhibitory effect of HPAC on cytokine secretion may be likely via NF-κB regulation. To elucidate the inhibitory effect of HPAC on NF-κB nuclear expression, RAW 264.7 cells were pretreated with the indicated concentrations (100 and 200 μg/mL) of HPAC for 2 h and then stimulated with LPS for 2 h. Results showed that the protein levels of NF-κB p65 in the nuclear fraction significantly increased in the LPS-treated group (40fold), whereas HPAC treatment inhibited the nuclear translocation of NF-κB p65 (Figures 3(a) and 3(b)). e nuclear expression of NF-κB p65 in HPAC-treated cells (200 μg/mL) showed a 0.7-fold change compared to LPStreated cells (Figure 3(b)).
NF-κB transcriptional activity is suppressed by a stable IκB/NF-κB complex whereas activated IκB kinase, by inflammatory stimulus, phosphorylates IκB leading to ubiquitination and its degradation [39]. To explore how HPAC prevents LPS-induced NF-κB activation, we investigated the inhibitory effect of HPAC on the phosphorylation of IκBα, which led to NF-κB activation. Our data show that LPS strongly phosphorylated IκBα, by a 19-fold change, compared to control. And HPAC treatment attenuates the increased phosphorylation of IκBα, by 0.8-and 0.3-fold, compared to LPS treatment (Figures 3(c) and 3(d)). ese results suggest that HPAC reduced NF-κB nuclear translocation by inhibiting IκBα phosphorylation.

HPAC Regulates NO and PGE2
Production. NO is a vital proinflammatory mediator, and excessive NO production is involved in the pathogenesis of many inflammatory diseases [40,41]. Activation of iNOS, mainly regulated by NF-κB, increases NO production [42,43]. erefore, we examined the inhibitory effect of HPAC on LPS-induced iNOS and NO levels in RAW 264.7 cells. As shown in Figures 4(a) and 4(b), iNOS  (9), and fatty acids ester (10,11). expression was remarkably elevated by LPS stimulation and HPAC significantly attenuated the protein expression of iNOS in a dose-dependent manner. Furthermore, HPAC achieved the strongest inhibition (0.04-fold) at 200 μg/mL, which was more significant than DXM (0.5-fold) (Figure 4(b)). Next, NO production was analyzed by measuring the accumulation of nitrites in the supernatants using Griess assay. HPAC treatment significantly inhibited LPS-induced NO production dosedependently (Figure 4(c)). PGE 2 has been implicated in various biological actions, such as pain sensation and inflammatory condition [44]. COX-2 and mPGES-1 are functionally coupled and considered as the primary enzymes for the inflammatory PGE 2 generation [45]. Although the positive control DXM showed significant inhibitory effects against COX-2 expression, HPAC had no effect in decreasing COX-2 and mPGES-1 expression (Figures 4(a), 4(d), and 4(e)). 15-PGDH is identified as a catabolizing enzyme that converts PGE 2 into its inactive product [45]. HPAC treatment showed a dose-dependent increase in 15-PGDH expression and the maximum effect was achieved at 200 μg/mL (1.6-fold) (Figures 4(a) and 4(f)). Taken together, these results indicate that the inhibitory effect of HPAC on PGE 2 production is mediated by 15-PGDH upregulation (Figure 4(g)).

HPAC Regulates HO-1 via MAPK Signaling.
HO-1 is an important component of the cellular defense against inflammation [46], we further examined whether HPAC could induce HO-1 expression. Our data show that by treating RAW 264.7 cells at a fixed concentration of 200 μg/mL, HO-1 was expressed as early as 2 h and continuously increased until 8 h, after which the expression was reduced (Figures 5(a) and 5(b)). Also, in accordance with the different concentrations of HPAC, HO-1 expression significantly increased dose-dependently ( Figures 5(c) and 5(d)). Next, we investigated the underlying mechanism of HO-1 induction by HPAC treatment. MAPK family signaling cascades have been reported to induce HO-1 expression [47][48][49]. erefore, in the current study, the effects of HPAC on phosphorylation levels of MAPKs including JNK, p38, and ERK were analyzed. While phosphorylation of p38, ERK, and JNK MAPKs increased by HPAC treatment, significance only occurred at the highest concentration ( Figures 5(e)-5(g)).

Discussion
Processing, also known as Paozhi in Chinese or Poje in Korean, is a traditional pharmaceutic method involving techniques such as stir-frying, stewing, boiling, and steaming [52]. Before clinical application, different processing techniques are used to reduce and prevent toxicity and to induce effectiveness via guidance and concentration [53]. Studies show that processing reduced cytotoxicity of Gardenia jasminoides Ellis. and Xanthium sibiricum Patr. extracts compared to raw extracts [29,54]. Both HPAC and AC treatment showed significant cytotoxicity at 400 and 500 μg/ mL, but HPAC was considerably less toxic (Supplementary Figure 1). Inflammation is a complex biological process regulated by inflammatory mediators such as TNF-α, IL-1β, IL-6, NO, and PGE 2 . e excessive production of proinflammatory cytokines is generally recognized to play key roles in the development of inflammatory diseases [55,56]. DXM is a potent synthetic corticosteroid that exhibits anti-inflammatory and immunosuppressive effects [36]. DXM is widely used for the treatment of pneumonia, bronchiolitis, and rheumatoid arthritis [57][58][59]. e DXM-treated group was considered as the positive control. Numerous studies have shown that crude extracts and compounds isolated from AC possess anti-inflammatory effects in different cell types [21,22,34,60]. In parallel, our data demonstrate that HPAC reduces inflammatory responses in LPS-activated macrophage cells.
To further verify the effect of HO-1 on the crosstalk between MAPK pathway and NF-κB pathway, we blocked the HO-1 activity using specific MAPK inhibitors. Despite the fact that all three inhibitors blocked HO-1 activity in HPAC-treated RAW 264.7 cells, the MAPK inhibitors vary in their effect to reverse the expression of HO-1 induced by HPAC in LPSchallenged cells. Both SB203580 and SP600125 achieved HO-1 downregulation and nuclear NF-κB p65 upregulation; however, PD98059 showed no effect (Figures 6(a)-6(e)). Although HO-1 was suppressed, SP600125 was not sufficient to induce phosphorylation-induced degradation of IκBα compared to SB203580 (Figures 6(a)-6(c)). Interestingly, one study suggests crosstalk between JNK and NF-κB pathway in TNF-α-stimulated HepG2 cells [78]. e JNK inhibitor SP600125 reduced HSP27 phosphorylation, which plays a crucial role in the binding ability of IKK with IκBα [78]. It may be possible that SP600125 interfered with IκBα phosphorylation. Even though IκBα phosphorylation and degradation are impaired by JNK inhibition, the increased NF-κB nuclear translocation remains unclear. Nonetheless, only the p38/HO-1 signaling pathway seems to be required for phosphorylation and degradation of IκBα. Our results indicate the importance of p38/HO-1 signaling pathway for HPAC-mediated regulation on IκBα/NF-κB complex under LPS inflammatory condition.

Conclusions
AC has been widely consumed as a dietary product in Asia. Since heat processing is used to promote drug effectiveness and safety in herbal medicine, we provide evidence that HPAC decreases inflammatory responses. Previous studies have shown that pure capillarisin (CAP) isolated from AC showed muscle protective effect through MAPK and NF-κB signaling [79]. HPAC exhibits similar effects but its antiinflammatory effect is through regulating IκBα phosphorylation and NF-κB activation via HO-1 induction. Moreover, p38 MAPK signaling is required for HO-1 activation by HPAC in LPS-treated RAW 264.7 cells. Also, it is known that aerial parts of AC and some of its coumarin and flavonoid derivatives show anti-inflammatory activity via 5-lipoxygenase (LOX) inhibition [80]. Interestingly in this study, inhibition of PGE 2 secretion was mediated through 15-PGDH upregulation and not through COX-2 inhibition. Regulatory effects of HPAC on IκBα/NF-κB complex and 15-PGDH may explain the anti-inflammatory activity and HPAC may serve as a candidate for developing anti-inflammatory agents.
ere have been reports to achieve optimal extraction temperatures for enhancing HPAC anti-inflammatory capacity [81]. Further in vivo studies and experiments regarding heat processing conditions may help better understand the (drug) mechanism of HPAC and aid its development as an anti-inflammatory agent.

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

Conflicts of Interest
e authors declare no conflicts of interest.