Anti-Inﬂammatory Effects of Abeliophyllum distichum Nakai (Cultivar Okhwang 1) Callus through Inhibition of PI3K/Akt, NF- κ B, and MAPK Signaling Pathways in Lipopolysaccharide-Induced Macrophages

: One of the Korean endemic plants, Abeliophyllum distichum Nakai (Oleaceae), contains acteoside, which is a glycoside exhibiting neuroprotective, anti-inﬂammation effects and antibacte-rial capacities. We conducted an investigation on the effects of the callus of A. distichum (cultivar Okhwang 1, CAO) on pro-inﬂammatory mediators released following nuclear factor- к B (NF- к B), phosphatidylinositol 3-kinase/Akt (PI3K-Akt) and mitogen-activated protein kinase (MAPK) signal activation in lipopolysaccharide (LPS)-induced RAW 264.7 cells. Immunoblotting was employed to ﬁnd out the expression of cyclooxygenase-2 (COX-2), inducible nitric oxide (iNOS), and activation of MAPK molecules, NF- κ B and Akt. Cytokines, COX-2, and iNOS gene expression were assessed using polymerase chain reaction techniques. Cytokines, COX-2, and iNOS gene expression were assessed using polymerase chain reaction techniques. High-performance liquid chromatography revealed that CAO was rich in acteoside and isoacteoside. As a result, CAO inhibited the generation of NO, cytokines, COX-2, and iNOS expression. Further, translocation to the nuclear of NF- κ B p65 and degradation of the inhibitor of NF- к B (I к B) were alleviated by suppressing phosphorylation. Additionally, CAO signiﬁcantly impacted MAPK pathway activation by potentially reducing phosphorylation of MAPKs. These results indicate that the anti-inﬂammatory effect of CAO is mediated via the inhibition of MAPK, PI3K/Akt, and NF- κ B signaling pathways, probably via glycosides, phenolics, and ﬂavonoids bioactivity derived from plants. CAO can serve as a potential anti-inﬂammatory agent, which alleviates inﬂammation factors and act through speciﬁc cell signaling pathways.


Introduction
Inflammation serves as a response to harmful stimuli. The stimuli are caused by injured cells, pathogens, and endogenous signals, which result in cellular healing to restore normal function [1]. These reactions may repair injured tissues and maintain homeostasis, which is regulated by numerous cytokines and inflammatory mediators, which includes the tumor necrosis factor (TNF)-α, nitric oxide (NO), and interleukin-6 (IL-6) [2,3]. Lipopolysaccharide (LPS), the main ligand of Toll-like receptor 4 (TLR4), disproportionately initiates the response of innate immunity, resulting in enhanced inflammatory states and causing cell death [4]. LPS activates macrophages to secrete pro-inflammatory cytokines and reactive oxidative species (ROS) [5,6]. Under these stimulations, the pro-inflammatory mediators, including NO and prostaglandin E2 (PGE2), are generated in large amounts by inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively [7]. COX-2 and iNOS are expressed in responses to various pro-inflammatory cytokines, which can contribute to growing tumors by relating them with angiogenesis and apoptosis [8][9][10][11][12]. The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is involved

Chemicals and Reagents
All chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Lou USA) unless otherwise specified. Acteoside and isoacteoside were purchased fr ma. Acetonitrile, chloroform, dimethyl sulfoxide (DMSO), ethyl acetate, methan petroleum ether (HPLC-grade) were purchased from Merck (Darmstadt, Ge DMEM, FBS, penicillin/streptomycin, and trypsin were purchased from Hyclone UT, USA). The antibodies were purchased from Abcam (Cambridge, UK), San Biotechnology (Dallas, TX, USA), and Cell Signaling Technology (Boston, MA, US

Callus Induction
To induce callus formation [36], a 1 cm 2 piece of leaf explants were placed rashige and Skoog (MS) medium (containing 0.9% agar and 4% saccharose, p supplemented with NAA and 2,4-D, which cultured at 25 °C for 14~21 days quently, a sufficient amount of calli were obtained through subculture in the sa dium ( Figure 1b).

Extraction and Fractionation
The callus from A. distichum (cultivar Okhwang 1, 100 g) were extracted w methanol using a sonicator for 3 days. The extracts were concentrated using a vacuum evaporator (EYELA, Shanghai, China), and then the extracts were fract using petroleum ether and ethyl acetate three times. The ethyl acetate fraction o from A. distichum (CAO, 254 mg) was in DMSO for the experiment.

HPLC Analysis
A Waters 2695 system equipped with the Waters 2996 Photodiode array (PDA, MA, USA) was used to analyze CAO. The separation was performed u Xbridge-C18 (250 × 4.6 mm, 5 µm). The mobile phase consisted of acetonitrile (so and water containing 1% acetic acid (solvent B). The sample injection volume wa The flow rate was 1.0 mL/min, for a total run time of 20 min. The system was run gradient program: 0-20 min: 90% B to 50% B. The peaks of interest were moni 190-380 nm using a PDA detector and compared with the standard substances side and isoacteoside).

Callus Induction
To induce callus formation [36], a 1 cm 2 piece of leaf explants were placed on Murashige and Skoog (MS) medium (containing 0.9% agar and 4% saccharose, pH 5.7), supplemented with NAA and 2,4-D, which cultured at 25 • C for 14~21 days. Subsequently, a sufficient amount of calli were obtained through subculture in the same medium ( Figure 1b).

Extraction and Fractionation
The callus from A. distichum (cultivar Okhwang 1, 100 g) were extracted with 80% methanol using a sonicator for 3 days. The extracts were concentrated using a rotary vacuum evaporator (EYELA, Shanghai, China), and then the extracts were fractionated using petroleum ether and ethyl acetate three times. The ethyl acetate fraction of callus from A. distichum (CAO, 254 mg) was in DMSO for the experiment.

HPLC Analysis
A Waters 2695 system equipped with the Waters 2996 Photodiode array detector (PDA, MA, USA) was used to analyze CAO. The separation was performed using an Xbridge-C18 (250 × 4.6 mm, 5 µm). The mobile phase consisted of acetonitrile (solvent A) and water containing 1% acetic acid (solvent B). The sample injection volume was 10 µL. The flow rate was 1.0 mL/min, for a total run time of 20 min. The system was run using a gradient program: 0-20 min: 90% B to 50% B. The peaks of interest were monitored at 190-380 nm using a PDA detector and compared with the standard substances (acteoside and isoacteoside).

Cell Viability
The effect of CAO on cell viability was measured using the MTS assay kit (Promega, Madison, WI, USA), following the manufacturer's protocols. RAW 264.7 cells were seeded in 96-well plates for 24 h in complete medium. The cells were treated with CAO extracts (12.5-400 µg/mL) for 24 h, then exposed to the reagent for 2 h at 37 • C and 5% CO 2 . Next, the reduction was measured using the ELx808 plate reader (Biotek, Korea) at 490 nm.

NO Assay
RAW 264.7 cells were seeded for 24 h. The cells were then treated with CAO for 2 h with 1 µg/mL of LPS. The media were collected for NO analysis. The NO concentrations were analyzed using the Griess reagent (Sigma). Supernatants and the Griess reagent were mixed for 10 min. The absorbance was spectrophotometrically measured using a HumanCorp Xma-3000PC at 540 nm.

RT-PCR
Total RNA was extracted from RAW 264.7 cells using Nucleo Spin ® RNA Plus (Macherey-Nagel, Düren, Germany), and cDNA was synthesized using Rever Tra Ace -α-(Toyobo, Osaka, Japan) following the manufacturer's protocol. PCR was performed using Quick Taq ® HS Dye Mix (Toyobo). The transcription levels were normalized to those of GAPDH.

Quantitative PCR Analysis
The cDNAs obtained in the previous experiment were used as templates for qPCR, employing the Quanti Tect ® SYBR Green PCR kit (Qiagen, Hilden, Germany). To quantify the mRNA expression, the qPCR analysis was performed using a 7500 Real-Time PCR system (Applied Biosystems, CA, USA). The primers were designed using the Primer Table 1. Sequences of primers used in the RT-PCR and RT-qPCR analysis.

Immunofluorescence
Immunofluorescence was performed to visualize localization of NF-κB, IκB-α, and phosphorylation of Akt upon LPS stimulation. RAW 264.7 cells seeded on glass coverslips were treated with the CAO extract for 24 h with 1 µg/mL of LPS. After treatment, cells were fixed with 4% paraformaldehyde (Biosesang) Next, the cells were blocked with 3% BSA for 1 h. The cells were then incubated with the appropriate primary antibodies overnight at 4 • C. The primary antibodies included an anti-NF-κB p65 antibody (1:500), an anti-IκB-α antibody (1:500), and a phosphospecific anti-Akt antibody (1:500). After, cells were incubated with the appropriate secondary antibodies at a 1:1000 dilution of an anti-mouse IgG (Alexa Fluor ® 488) polyclonal antibody (ab150113) and an anti-rabbit IgG (Alexa Fluor ® 568) polyclonal antibody (ab175471, Abcam) or 300 nM of DAPI (D1306, Invitrogen, Waltham, MA, USA) for 5 min in the dark. Finally, sample coverslips were mounted using Fluorescence Mounting Medium (S3023, Dako, Carpinteria, CA, USA). The observations were performed using a fluorescence microscope and a CKX53 microscope (Olympus, Tokyo, Japan) with 400× magnification, and micrographs were captured with a Digital Single-Lens Reflex Camera (DS126271, Canon, Tokyo, Japan).

Statistical Analysis
All the data were analyzed using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) and are presented as the mean ± standard deviation. The data were analyzed using the one-way analysis of variance, with Tukey's multiple-comparisons post-hoc test employed for comparing mean values among multiple groups.

Analysis of Acteoside and Isoacteoside in CAO
To identify and quantify acteoside and isoacteoside of CAO, the major chemical constituents were analyzed by HPLC and compared with its standards. The chromatogram of CAO was identified by comparing the retention time (RT) to that of standard acteoside and isoacteoside at 330 nm. The HPLC results revealed acteoside and isoacteoside contained in CAO (RT = 19.87 and 22.85) (Figure 2A), and it was compared with that of standard acteoside and isoacteoside (RT = 20.10 and 22.83) ( Figure 2B). Compared with the standard curve, the amount of acteoside was analyzed to be 399.27 ± 2.3 mg/g (39.93% w/w), and isoacteoside was analyzed to be 56.69 ± 1.1 mg/g (5.67% (w/w) in CAO. These results confirmed that acteoside and isoacteoside, with chemical structures shown in Figure 2C,D, are the major compounds in CAO.  Figure 2B). Compared with the standard curve, the amount of acteoside was analyzed to be 399.27 ± 2.3 mg/g (39.93% w/w), and isoacteoside was analyzed to be 56.69 ± 1.1 mg/g (5.67% (w/w) in CAO. These results confirmed that acteoside and isoacteoside, with chemical structures shown in Figure 2C,D, are the major compounds in CAO.

Effects of CAO on Cell Viability
To conduct cell viability assay, RAW 264.7 macrophages were cultured with CAO (0-400 µg/mL). After 24 h, cell viability was assayed using MTS assay. In macrophages, the CAO was non-cytotoxic below 400 µg/mL and did not significantly suppress cell growth. The results demonstrated that the CAO extract was non-cytotoxic in RAW 264.7 cells below 400 µg/mL and did not significantly suppress cell growth ( Figure 3A).

Effects of CAO on Cell Viability
To conduct cell viability assay, RAW 264.7 macrophages were cultured with CAO (0-400 µg/mL). After 24 h, cell viability was assayed using MTS assay. In macrophages, the CAO was non-cytotoxic below 400 µg/mL and did not significantly suppress cell growth. The results demonstrated that the CAO extract was non-cytotoxic in RAW 264.7 cells below 400 µg/mL and did not significantly suppress cell growth ( Figure 3A).
(39.93% w/w), and isoacteoside was analyzed to be 56.69 ± 1.1 mg/g (5.67% (w/w) in CAO. These results confirmed that acteoside and isoacteoside, with chemical structures shown in Figure 2C,D, are the major compounds in CAO.

Effects of CAO on Cell Viability
To conduct cell viability assay, RAW 264.7 macrophages were cultured with CAO (0-400 µg/mL). After 24 h, cell viability was assayed using MTS assay. In macrophages, the CAO was non-cytotoxic below 400 µg/mL and did not significantly suppress cell growth. The results demonstrated that the CAO extract was non-cytotoxic in RAW 264.7 cells below 400 µg/mL and did not significantly suppress cell growth ( Figure 3A).

Effects of CAO on Pro-Inflammatory Cytokine Secretions
As shown in Figure 3B, the levels of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) increased under LPS treatment. However, treatments with CAO (25 and 50 µg/mL) showed inhibitory effects against LPS-induced pro-inflammatory cytokine se-Processes 2021, 9, 1071 7 of 14 cretions in macrophages. In particular, CAO at 50 µg/mL significantly down-regulated the released levels of pro-inflammatory cytokines (IL-6 and IL-1β) compared with the LPS-treated group.

Effects of CAO on IκB/NF-κB Signaling Pathways
To demonstrate the effects of CAO on the signaling pathway of IκB/NF-κB, focuses on the inhibited inflammatory response, immunoblotting was carried out to confirm the inhibitory effect of CAO on the phosphorylation of NF-κB and degradation of IκB. Our results demonstrated that CAO suppresses the NF-κB signaling mechanisms by inhibiting the phosphorylation of IκB-α. CAO significantly blocked the degradation of IκB-α in a concentration-dependent manner. Furthermore, CAO significantly attenuated p-p65 without altering the total level of p65 ( Figure 5B,D). Next, we performed an immunofluorescence investigation to monitor the expression of the NF-κB subunit. Immunofluorescence data obtained from individual cells confirmed our finding that CAO treatment at 50 µg/mL potentially inhibited the translocation of phosphorylated NF-κB (subunit p65) and the degradation of IκB-α ( Figure 6A,B).

Effects of CAO on IκB/NF-κB Signaling Pathways
To demonstrate the effects of CAO on the signaling pathway of IκB/NF-κB, focuses on the inhibited inflammatory response, immunoblotting was carried out to confirm the inhibitory effect of CAO on the phosphorylation of NF-κB and degradation of IκB. Our results demonstrated that CAO suppresses the NF-κB signaling mechanisms by inhibiting the phosphorylation of IκB-α. CAO significantly blocked the degradation of IκB-α in a concentration-dependent manner. Furthermore, CAO significantly attenuated p-p65 without altering the total level of p65 ( Figure 5B,D). Next, we performed an immunofluorescence investigation to monitor the expression of the NF-κB subunit. Immunofluorescence data obtained from individual cells confirmed our finding that CAO treatment at 50 µg/mL potentially inhibited the translocation of phosphorylated NF-κB (subunit p65) and the degradation of IκB-α ( Figure 6A,B).

Effects of CAO on PI3K-Akt Activation and MAPKs Pathway
The activation of the MAPK (ERK1/2, JNK1/2, and p38) and PI3K-Akt signaling pathway is crucial for initiating the NF-κB signal transduction pathway. We sought to identify the activities of action by which CAO reduces the production of inflammatory cytokines. LPS induction significantly increased Akt phosphorylation; this enhanced phosphorylation was suppressed by CAO. Furthermore, CAO demonstrated that the ERK1/2 (except 25 µg/mL), JNK1/2, and p38 phosphorylation levels induced by LPS were significantly inhibited ( Figure 5A,C). Additionally, we confirmed the effect of CAO on the phosphorylation of Akt through immunoblotting ( Figure 5B) and immunofluorescence investigations ( Figure 6A). These results demonstrated that CAO suppressed the expression and detection of phospho-Akt in individual cells. Collectively, our results suggest that CAO reduced the production of NO, COX-2, and iNOS expression by down-regulating the LPS-induced ERK, JNK, p38 MAPKs, IκB-α/NF-κB, and PI3K/Akt signaling pathways.

Effects of CAO on PI3K-Akt Activation and MAPKs Pathway
The activation of the MAPK (ERK1/2, JNK1/2, and p38) and PI3K-Akt signaling pathway is crucial for initiating the NF-κB signal transduction pathway. We sought to identify the activities of action by which CAO reduces the production of inflammatory cytokines. LPS induction significantly increased Akt phosphorylation; this enhanced phosphorylation was suppressed by CAO. Furthermore, CAO demonstrated that the ERK1/2 (except 25 µg/mL), JNK1/2, and p38 phosphorylation levels induced by LPS were significantly inhibited ( Figure 5A,C). Additionally, we confirmed the effect of CAO on the phosphorylation of Akt through immunoblotting ( Figure 5B) and immunofluorescence investigations ( Figure 6A). These results demonstrated that CAO suppressed the expression and detection of phospho-Akt in individual cells. Collectively, our results suggest that CAO reduced the production of NO, COX-2, and iNOS expression by down-regulating the LPS-induced ERK, JNK, p38 MAPKs, IκB-α/NF-κB, and PI3K/Akt signaling pathways.

Discussion
According to a study on chemotaxonomy of the Oleaceae [37], the Oleaceae synthesizes and distributes iridoid glucosides. Abeliophyllum, one of the Oleace ily, synthesizes caffeoyl phenylethanoid glycosides (cornoside and acteoside). In ular, A. distichum Nakai is known to predominantly contain acteoside and isoac [24]. Regarding A. distichum, which consists of only one species worldwide, sev searchers have investigated its environmental and ecological characteristics, g sequences, and bioactivities [21,22,[38][39][40][41]. Reportedly, a previous study has prov the inhibitory effects on the inflammation of the A. distichum leaf are facilitated inhibition of ERK-mediated NF-κB phosphorylation in mouse monocytes [42]. Ho research on Okhwang 1, a new official cultivar of A. distichum, is lacking, exc studies regarding the genetic characteristics. Moreover, the study on the pharma cal properties of the callus induced by plant cell culture remains insufficient. In t sent study, we tried to assess whether the treatment of RAW 264.7 macrophag CAO may affect the expression on selected pro-inflammatory factors. To analy chemicals present in CAO, HPLC analyses were performed to identify major chemicals. As a result of HPLC analysis, two components are predominant in which are analyzed to acteoside and isoacteoside compared to purchased st products. Macrophages/monocytes can be stimulated by LPS to p pro-inflammatory molecules (NO, PGE2, IL-1β, TNF-α, IL-6, etc.) by leading sig pathways (including IκB/NF-κB, PI3K/Akt, and MAPKs) in cells [43][44][45]. In the cesses, the immune system adjusts macrophages, which play a crucial role in resp immune or participating in inflammatory responses [46][47][48][49]. COX-2 and iNOS portant factors involved in the production of excess PGE2 and NO at the inflam sites and are known to play important roles during the pathogenesis of chronic d

Discussion
According to a study on chemotaxonomy of the Oleaceae [37], the Oleaceae family synthesizes and distributes iridoid glucosides. Abeliophyllum, one of the Oleaceae family, synthesizes caffeoyl phenylethanoid glycosides (cornoside and acteoside). In particular, A. distichum Nakai is known to predominantly contain acteoside and isoacteoside [24]. Regarding A. distichum, which consists of only one species worldwide, several researchers have investigated its environmental and ecological characteristics, genome sequences, and bioactivities [21,22,[38][39][40][41]. Reportedly, a previous study has proved that the inhibitory effects on the inflammation of the A. distichum leaf are facilitated via the inhibition of ERK-mediated NF-κB phosphorylation in mouse monocytes [42]. However, research on Okhwang 1, a new official cultivar of A. distichum, is lacking, except for studies regarding the genetic characteristics. Moreover, the study on the pharmacological properties of the callus induced by plant cell culture remains insufficient. In the present study, we tried to assess whether the treatment of RAW 264.7 macrophages with CAO may affect the expression on selected pro-inflammatory factors. To analyze the chemicals present in CAO, HPLC analyses were performed to identify major phytochemicals. As a result of HPLC analysis, two components are predominant in CAO, which are analyzed to acteoside and isoacteoside compared to purchased standard products. Macrophages/monocytes can be stimulated by LPS to produce pro-inflammatory molecules (NO, PGE2, IL-1β, TNF-α, IL-6, etc.) by leading signaling pathways (including IκB/NF-κB, PI3K/Akt, and MAPKs) in cells [43][44][45]. In these processes, the immune system adjusts macrophages, which play a crucial role in responding immune or participating in inflammatory responses [46][47][48][49]. COX-2 and iNOS are important factors involved in the production of excess PGE2 and NO at the inflammatory sites and are known to play important roles during the pathogenesis of chronic diseases [47,50,51]. Therefore, the strong inhibitory effects on iNOS and COX-2 expression, documented by several reports, are immensely worthy in treating and preventing inflammatory disorders [49]. Results of cell viability indicate that CAO did not significantly affect cell proliferation. In LPS-induced macrophages, CAO significantly inhibited producing NO. NO production levels are related to the expression of COX-2 and iNOS. CAO significantly inhibited LPS-induced COX-2 and iNOS expression at the transcriptional levels. These findings suggest that the suppression of NO release by CAO could be due to the inhibition of COX-2 and iNOS. MAPKs are involved in directing cellular responses to pro-inflammatory cytokines. Additionally, they control cellular functions, including gene expression, apoptosis, proliferation, mitosis, differentiation, and cell survival [52]. The MAPKs, which control cellular signal transduction to the nucleus, consist of serine/threonine kinases, such as JNK, p38 MAPK, and ERK [53]. The phosphorylation of MAPKs has been involved in signaling pathways linked to LPS-induced inflammation, which suggests that MAPKs are a major molecular for regulating inflammation [54,55]. We identified the mechanism of action by which CAO reduces the production of inflammatory mediators. CAO significantly inhibited LPS-induced ERK, JNK, and p38 MAPKs. The ability of CAO to regulate the phosphorylation of ERK1/2, JNK, and p38 may also affect a reduction in COX-2 and iNOS expression. These results are supported by the fact that MAPKs are involved in the regulation of COX-2 and iNOS genes, as inhibition of MAPK activity results in the suppression of COX-2 and iNOS gene expression [56]. Based on the inhibitory effects of CAO on COX-2 and iNOS expression, we investigated whether CAO could suppress the phosphorylation IκB/NF-κB pathway under LPS stimulation. Furthermore, we confirmed the expression of NF-κB by an immunofluorescence investigation. NF-κB that controls the transcription of DNA regulates the expression of target genes coding for pro-inflammatory mediators involved in immune and inflammatory responses, including cytokines [57,58]. Therefore, NF-κB is regarded as a target molecular for inflammatory treatment strategies [59,60]. The degradation of IκB and phosphorylation of MAPKs are the upstream regulatory signaling pathways for the transcriptional activation of NF-κB [13]. CAO reduced LPS-stimulated inflammation through the inhibition of phosphorylation of NF-κB. Furthermore, phosphorylation and degradation of IκB were inhibited following CAO treatment. Based on immunofluorescence data detected in individual cells, we confirmed our discovery that the LPS-induced phosphorylation and translocation to the nucleus of NF-κB was potentially inhibited by CAO treatment. These findings verify that CAO might regulate the production of cytokines, chemokines, and NO through suppressing IκB degradation, thus inhibiting translocation to the nucleus of NF-κB. Therefore, the inhibition of these signaling pathways could demonstrate the potential of CAO as a suppressor of inflammatory mediators. Additionally, IκB/NF-κB, MAPKs, and PI3K/Akt is another signaling pathway that controls the expression of inflammatory factors by activating the NF-κB signal transduction pathway [61]. Thus, suppression of phosphorylation is known to be a main target to modulate inflammatory disorders. CAO significantly suppressed LPS-induced PI3K/Akt phosphorylation. Furthermore, immunofluorescence data from cells confirmed that the phosphorylation of Akt was inhibited by CAO treatment, leading to the retention of p65 in the cytoplasm. Additionally, correlation among the expression of IκB and the penetration to the nuclear of p65 was shown. In the present study, we demonstrated that the suppression of NO production and iNOS and COX-2 expression by CAO was mediated through the down-regulation of MAPKs, NF-κB, and PI3K/Akt pathways. In conclusion, we suggest that CAO has anti-inflammatory effects and could be a superior candidate for an anti-inflammation agent derived from natural resources.

Conclusions
In summary, treatment of CAO can reduce the levels of cytokines, iNOS, and COX-2 in RAW 264.7 macrophages. Moreover, CAO affected particular proteins regulating MAPK, NF-κB, and PI3K-Akt signaling pathways. In conclusion, the present study suggests that the treatment of CAO could be a potential resource for alleviating inflammatory diseases by significantly attenuating specific factors in LPS-induced signaling pathways.