Anti-Inflammatory Effects of Angelica sinensis (Oliv.) Diels Water Extract on RAW 264.7 Induced with Lipopolysaccharide

The dry root of Angelica sinensis (Oliv.) Diels, also known as “female ginseng”, is a popular herbal drug amongst women, used to treat a variety of health issues and cardiovascular diseases. The aim of this study is to evaluate the detailed molecular mechanism for anti-inflammatory effects of Angelica sinensis root water extract (ASW). The anti-inflammatory effect of ASW on lipopolysaccharide (LPS)-induced RAW 264.7 mouse macrophages was evaluated by the tetrazolium-based colorimetric assay (MTT), Griess reagent assay, multiplex cytokine assay, real time reverse transcription polymerase chain reaction (RT-PCR), and Fluo-4 calcium assay. ASW restored cell viability in RAW 264.7 at concentrations of up to 200 µg/mL. ASW showed notable anti-inflammatory effects. ASW exhibited IC50 = 954.3, 387.3, 191.7, 317.8, 1267.0, 347.0, 110.1, 573.6, 1171.0, 732.6, 980.8, 125.0, and 257.0 µg/mL for interleukin (IL)-6, tumor necrosis factor (TNF)-α, monocyte chemotactic activating factor (MCP)-1, regulated on activation, normal T cell expressed and secreted (RANTES), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), lipopolysaccharide-induced CXC chemokine (LIX), macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, IL-10, and intracellular calcium, respectively. Additionally, ASW inhibited the LPS-induced production of nitric oxide and the LPS-induced mRNA expression of CHOP (GADD153), Janus kinase 2 (JAK2), signal transducers and activators of transcription 1 (STAT1), first apoptosis signal receptor (FAS), and c-Fos, NOS2, and PTGS2 (COX2) in RAW 264.7 significantly (p < 0.05). Data suggest that ASW exerts an anti-inflammatory effect on LPS-induced RAW 264.7 via NO-bursting/calcium-mediated JAK-STAT pathway.


Introduction
Immunity is a core function of human life against pathologic infections. It is our first-defense mechanism against infection as an innate immune activity. Innate immune activity includes inflammasome and pyroptosis [1]. Inflammasome (pyroptosome) provokes cytokine production and leads to programmed cell death-pyroptosis. Pyroptosis, distinct from apoptosis, is a programmed, immune cell death caused by intracellular pathogens [1]. Inflammation is indispensable during an immune reaction against pathogens, such as bacteria, viruses, and fungi. With inflammation, we experience heat, pain, edema, and redness. Consequently, inflammatory mediators can be produced massively against infectious pathogens, which may be hazardous to the human host. Thus, it is important to both remove pathogens, and modulate hyper-inflammatory activity and excessive production of inflammatory mediators, such as nitric oxide (NO) and cytokines.
NO, which can act as a free radical, is not only a strong toxic molecule to combat invasive pathogens (bacteria, virus, etc.), but also a powerful vasodilator in the cellular signaling pathway of inflammation. NO is regarded as playing a major role in the fatal hypotension of septic shock and sepsis.
Cytokines, including chemokines and growth factors, also play an important role in hypersensitive reactions against both exogenous and endogenous antigens. Cytokines may be maturated by an inflammasome-promoted process.
In 2011, Chao et al. reported that Angelica sinensis (Oliv.) Diels ethyl acetate extract significantly inhibited NF-kappaB trans-activation activity with down-regulating NO and cytokines from lipopolysaccharide (LPS), plus IFN-γ-induced RAW 264.7 [2]. However, the effect of Angelica sinensis root water extract (ASW) on LPS-induced macrophages to our knowledge has not been reported.
In the present study, we investigated the anti-inflammatory effects of ASW using multiplex cytokine assay and quantitative real time reverse transcription polymerase chain reaction (RT-PCR) in lipopolysaccharide (LPS)-induced RAW 264.7 mouse macrophages. The resulting data suggests that ASW exerts anti-inflammatory effects via NO-bursting/calcium-mediated Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway.

Reagents
Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco BRL (Grand Island, NY, USA) and all other chemicals for the cell culture were obtained from Merck Millipore (Darmstadt, Germany).

Preparation of ASW
The commercial product of Angelica sinensis root was obtained from Omniherb (Daegu, Korea) and authenticated by Professor Y. J. Lee of Gachon University in June 2015. A voucher specimen (No. 2015-0027) was deposited at the Department of Pathology in Gachon University College of Korean Medicine. Because herbal drugs have been traditionally extracted using water, in the present study, Angelica sinensis root was extracted with boiling water for 2 h, filtered, and then lyophilized (yield: 50.63%). The powdered extract was dissolved in saline and then filtered through a 0.22 µm syringe filter.

Total Flavonoid Content
The total flavonoid content of ASW was determined using the diethylene glycol colorimetric method. Briefly, the sample solution (20 µL of 2 mg/mL ASW) was mixed with 200 µL of diethylene glycol and 20 µL of 1 N NaOH. The sample absorbance was read at 405 nm after 1 h incubation at 37 • C. Rutin was used as a reference standard and the total flavonoid content was expressed as milligrams of rutin equivalents (mg RE/g extract) [3].

Cell Viability
RAW 264.7 were obtained from Korea Cell Line Bank (Seoul, Korea). RAW 264.7 (passage number 7) were cultured with DMEM supplemented with 10% FBS containing 100 U/mL of penicillin and 100 µg/mL of streptomycin at 37 • C in a 5% CO 2 humidified incubator according to the protocol of previous study [4]. Before experimental assays, RAW 264.7 were washed with phosphate buffer saline. Briefly, RAW 264.7 were cultured with samples for 24 h in 96-well plates (each well containing 10,000 cells) in order to verify the toxicity of ASW. After 24 h culture with samples, cell viability was confirmed with the tetrazolium-based colorimetric assay (MTT) according to the protocol of a previous study [4].

NO Concentration
After 24-h incubation with samples in 96-well plates, NO levels in each well containing 10,000 cells were identified using the Griess reagent according to a previous study [4].

Intracellular Calcium Assay
Fluo-4 AM is a well-known fluorescent Ca 2+ indicator used for in-cell measurement of calcium signaling. After 18 h treatment with samples in 96-well plates, the intracellular signaling from each well containing 100,000 cells was identified using Fluo-4 NW Calcium Assay Kits (Thermo Fisher Scientific, Waltham, MA, USA) according the protocol of our previous study [5][6][7]. The assay was done with a spectrofluorometer (Dynex, West Sussex, UK) with excitation and emission filters of 485 nm and 535 nm, respectively. Indomethacin, which is known to impair mitochondrial calcium uptake, was used as a positive control.

Statistical Analysis
Experimental results are presented as mean ± standard deviation. Experiments were done more than three times. Data analysis was performed by Student t-test or one-way analysis of variance test followed by Tukey's multiple comparison test, as appropriate. For statistics, the program Graphpad Prism 4.01 (Graphpad Software, La Jolla, CA, USA) was used. A p-value < 0.05 was considered statistically.

Determination of the Total Flavonoid Content of ASW
Our data represented that the total flavonoid content of ASW was 5.14 mg RE/g extract.

Discussion
These days, researchers have understandably started focusing on developing immuno-modulatory therapies and anti-inflammatory drugs that deal with inflammasome inhibition, pyroptosis suppression, toll-like receptor block, transcript factor deactivation, phosphoprotein dephosphorylation, mitogen-activated protein kinases deactivation, and so on. Moreover, studies on many herbal drugs and natural products have reported them to have anti-inflammatory effects without cytotoxicity [8][9][10].
For example, a study by Yoon et al. reported that Scutellaria baicalensis Georgi water extract inhibited LPS-induced production of NO, IL-3, IL-6, IL-10, IL-12p40, IL-17, IP-10, keratinocyte-derived chemokine (KC), and VEGF in mouse macrophages [11]. A study by Yuk et al. similarly reported that Epimedium brevicornum Maxim water extract inhibits LPS-induced production of NO, IL-3, IL-10, IL-12p40, IP-10, KC, VEGF, MCP-1, and GM-CSF in mouse macrophages [12]. These studies notably found that these herbal drugs did not present any cytotoxicity on macrophages because pyroptosis (the inflammatory programmed cell death, in which immune cells release pro-inflammatory cytokines, swell, burst, and die) is a distinct aspect of infection-induced intracellular inflammatory pathway in immune cells. Like Scutellaria baicalensis water extract and Epimedium brevicornum water extract, ASW (up to a concentration of 200 µg/mL) did not show any cytotoxic effect on macrophages in the present study. In addition to the anti-inflammatory effect of Angelica sinensis ethyl acetate extract on LPS, plus IFN-γ-induced RAW 264.7 [2]. Su et al. reported that ligustilide, a major compound of Angelica sinensis root, significantly suppressed NO, TNF-α, and prostaglandin E2 production from LPS-induced RAW 264.7 by deactivation of MAPK, NF-kappaB, and AP-1 [13]. Interestingly, our data represented that ASW inhibits excessive release of intracellular calcium as well as overproduction of NO and cytokines in LPS-induced RAW 264.7. However, it is a limitation that IL-10, a well-known anti-inflammatory cytokine, was decreased by ASW in the present assay. It is another shortcoming that this study could not evaluate the effect of ASW on mitogen-activated protein kinase cascade caused by LPS.
LPS-induced macrophages release many kinds of inflammatory mediators such as NO, interleukins, chemokines, growth factors, and so forth. Thus, LPS-stimulated mouse macrophage is a common model for examining the anti-inflammatory effect of natural products and herbal medicines. This is because LPS causes an endoplasmic reticulum (ER) stress with the overexpression of CHOP, which initiates inflammasome activation and subsequent macrophage pyroptosis [14].
Interestingly, although originally produced to fight against infectious pathogens, NO plays a role as an ER stress and brings about an unfolded protein response. In conjunction, as an effector of the unfolded protein response, CHOP amplifies the release of ER calcium store and activates STAT pathway [15][16][17]. Additionally, the JAK-STAT pathway has been already reported to play a critical role in the proinflammatory gene expression of RAW 264.7 [18].
Thus, it is meaningful to examine the effects of ASW on LPS-induced CHOP overexpression and pyroptosis signaling. In the present study, our experimental data showed that Angelica sinensis water extract inhibits the overexpression of CHOP, JAK2, STAT1, FAS, c-Fos, NOS2, and PTGS2 in LPS-induced macrophages.
We also found it pertinent to evaluate intracellular calcium. It is an important signaling molecule of ER stress and increases with CHOP overexpression by LPS induction.
Finally, we found that ASW restored the cell viability in LPS-induced RAW 264.7 at concentrations of up to 200 µg/mL. ASW showed notable anti-inflammatory effects at these concentrations. Data revealed that ASW exhibited IC 50 = 954.

Conflicts of Interest:
The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.