Retrofractamide C Derived from Piper longum Alleviates Xylene-Induced Mouse Ear Edema and Inhibits Phosphorylation of ERK and NF-κB in LPS-Induced J774A.1

Many studies have reported the biological activities of retrofractamide C (RAC). However, few studies have investigated the anti-inflammatory effect of RAC. In the present study, we investigated the anti-inflammatory effect of RAC using lipopolysaccharide (LPS)-induced J774A.1 cells and a xylene-induced mouse ear edema model. Treatment with RAC decreased LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) secretion and inducible NO synthase (iNOS) and cyclooxygenase 2 (COX2) protein expression. It also downregulated the LPS-induced production of interleukin-1β (IL-1β) and interleukin-6 (IL-6) but not tumor necrosis factor α (TNF-α). In the LPS-induced signaling pathway, RAC inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) and nuclear factor kappa light chain enhancer of activated B cells (NF-κB) but not c-Jun N-terminal kinase (JNK) or p38. In a xylene-induced mouse ear edema model, RAC treatment alleviated edema formation and inflammatory cell infiltration. In conclusion, the present study indicates that RAC has the potential to have anti-inflammatory effects and could be a prospective functional food.


Introduction
The role of the inflammatory response is to defend the body against infection and tissue injury [1][2][3]. The inflammatory response is initiated by recognition of pathogen-associated molecular patterns (PAMPs) of foreign substances or damage-associated molecular patterns (DAMPs) of injured tissue by pattern recognition receptors (PRRs) of residing or circulating immune cells, such as macrophages, mast cells, fibroblasts and leukocytes [3,4]. Toll-like receptor (TLR) family members are PRRs that detect various molecules, such as extracellular components and nucleic acids of bacteria and viruses, according to their subtype [5,6]. Once a PRR binds its agonist, downstream signaling pathways, including mitogen-activated protein kinases (MAPKs) and the nuclear factor kappa light chain enhancer

Results and Discussion
2.1. RAC Inhibits NO and PGE2 Production in LPS-Induced J774A.1 Cells NO and PGE2 are major mediators of the inflammatory response. Their biosynthesis is significantly upregulated in inflamed regions, and they contribute to the pathogenesis of inflammatory disorders [30,31]. NO is produced by NO synthase (NOS) and converted from L-arginine in the presence of NADPH and oxygen. In this process, tetrahydrobiopterin (BH4), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and heme act as a cofactor [32]. PGE2 synthesis is initiated with arachidonic acid formation from phospholipids of the cell membrane by phospholipase A2 (PLA2). Arachidonic acid is converted to prostaglandin H 2 (PGH2) by cyclooxygenase (COX) then, PGH2 is converted to PGE2 by PGE2 synthase [33]. NO and PGE productions are regulated by substrates availability, enzyme expressions and enzyme activities. Previous studies showed that reducing NO and PGE2 levels could reduce the symptoms of inflammatory diseases [30][31][32][33][34]. Before investigating the anti-inflammatory effect of RAC ( Figure 1A), an MTT assay for the cytotoxicity of RAC was performed. There was no cytotoxicity on J774A.1 cells at the indicated concentration of RAC ( Figure 1B). After that, we investigated the effect of RAC on LPS-induced NO and PGE2 production to evaluate the anti-inflammatory effect of RAC. RAC treatment inhibited both NO and PGE2 production in a dose-dependent manner ( Figure 1C, D). LPS-induced NO and PGE2 production to evaluate the anti-inflammatory effect of RAC. RAC treatment inhibited both NO and PGE2 production in a dose-dependent manner ( Figure 1C, D). h. (C) The secretion of NO was measured by an NO assay. (D) PGE2 production was determined by ELISA. (E) iNOS and COX2 protein expression decreased by RAC treatment. J774A.1 cells were treated with 200 ng/mL LPS for 18 h after treatment with dexamethasone and RAC for 1 h. The iNOS and COX2 protein expression levels were determined by immunoblot assay. The band optical densities were calculated by ImageJ software. Representative data are presented. Values are presented as the mean ± SD of three individual experiments. * p < 0.05, ** p < 0.01 compared with the LPS only-treated group. DEX, 10 μM dexamethasone.

RAC Decreases iNOS and COX2 Expression in LPS-Induced J774A.1 Cells
The synthesis of NO and PGE2 is achieved by NOS and COX enzymes. They have several isotype enzymes; however, specific enzymes, iNOS and COX2, are responsible for NO and PGE2 production

RAC Decreases iNOS and COX2 Expression in LPS-Induced J774A.1 Cells
The synthesis of NO and PGE2 is achieved by NOS and COX enzymes. They have several isotype enzymes; however, specific enzymes, iNOS and COX2, are responsible for NO and PGE2 production in the inflammatory response [30][31][32]. Inhibition of their activity and expression reduces NO and PGE2 levels and could alleviate inflammatory disorders [31,32]. To determine whether RAC affected LPS-induced iNOS and COX2 protein expressions, immunoblot assay was performed. iNOS expression was significantly decreased after 3 and 10 µM RAC treatment, and COX2 expression was significantly downregulated after 10 µM RAC treatment ( Figure 1E). There are several factors, which regulated both iNOS and COX2 expressions, including transforming growth factor β (TGF-β), NF-κB and AP-1. In macrophage cells, TGF-β inhibits COX2 expression and it increases degradation of iNOS and decreases iNOS mRNA stability [32,33]. The expressions of iNOS and COX2 are also regulated by NF-κB and AP-1, which are transcriptional factors. Thus, RAC treatment could affect these factors and downregulation of iNOS and COX2 protein expressions.
2.3. RAC Inhibits IL-1β and IL-6 But Not TNF-α Gene Expression in LPS-Induced J774A.1 Cells Proinflammatory cytokines induced by activated innate immune cells mediate and amplify the inflammatory response through activation of their downstream signaling cascades [34,35]. They are also related to the pathogenesis and hyperalgesia of inflammatory disorders [36][37][38]. The effect of RAC on proinflammatory cytokine expression was evaluated by quantitative real-time PCR. As shown, LPS-induced IL-1β and IL-6 expression was significantly decreased after 3 and 10 µM RAC treatment, but TNF-α expression was not affected ( Figure 2). Regulation of these cytokine gene expressions is affected by various factors. LPS stimulation leads to activation of transcription factors such as NF-κB, AP-1, IRF and CCAAT/enhancer-binding protein β (C/EBPβ) and they promote proinflammatory cytokine gene expressions [39]. The cytokine mRNA is controlled by post-transcriptional regulation. Binding of RNA binding proteins or microRNA with cytokine mRNA decreases RNA stability and increases mRNA degradations [40].
Molecules 2020, 25, x FOR PEER REVIEW 4 of 11 in the inflammatory response [30][31][32]. Inhibition of their activity and expression reduces NO and PGE2 levels and could alleviate inflammatory disorders [31,32]. To determine whether RAC affected LPS-induced iNOS and COX2 protein expressions, immunoblot assay was performed. iNOS expression was significantly decreased after 3 and 10 μM RAC treatment, and COX2 expression was significantly downregulated after 10 μM RAC treatment ( Figure 1E). There are several factors, which regulated both iNOS and COX2 expressions, including transforming growth factor β (TGF-β), NF-κB and AP-1. In macrophage cells, TGF-β inhibits COX2 expression and it increases degradation of iNOS and decreases iNOS mRNA stability [32,33]. The expressions of iNOS and COX2 are also regulated by NF-κB and AP-1, which are transcriptional factors. Thus, RAC treatment could affect these factors and downregulation of iNOS and COX2 protein expressions.

RAC Inhibits IL-1β and IL-6 But Not TNF-α Gene Expression in LPS-Induced J774A.1 Cells
Proinflammatory cytokines induced by activated innate immune cells mediate and amplify the inflammatory response through activation of their downstream signaling cascades [34,35]. They are also related to the pathogenesis and hyperalgesia of inflammatory disorders [36][37][38]. The effect of RAC on proinflammatory cytokine expression was evaluated by quantitative real-time PCR. As shown, LPS-induced IL-1β and IL-6 expression was significantly decreased after 3 and 10 μM RAC treatment, but TNF-α expression was not affected (Figure 2). Regulation of these cytokine gene expressions is affected by various factors. LPS stimulation leads to activation of transcription factors such as NF-κB, AP-1, IRF and CCAAT/enhancer-binding protein β (C/EBPβ) and they promote proinflammatory cytokine gene expressions [39]. The cytokine mRNA is controlled by posttranscriptional regulation. Binding of RNA binding proteins or microRNA with cytokine mRNA decreases RNA stability and increases mRNA degradations [40].  MAPKs and NF-κB signaling are involved in the TLR family signaling pathway, as part of the downstream signaling cascade [5,6]. Inhibition of these signaling molecules is a good strategy to treat inflammatory diseases. To investigate the effect of RAC on MAPKs and NF-κB signaling molecules, immunoblot analysis was performed. The results showed that RAC treatment downregulated the phosphorylation of ERK and NF-κB p65 ( Figure 3A,B). However, it did not affect the phosphorylation of JNK and p38 ( Figure 3A). These results indicate that RAC selectively regulates signaling molecules, ERK and NF-κB. There are some studies showing that selective inhibition of ERK and NF-κB could not affect the production of TNF-α but decreased other inflammatory mediators, such as IL-1β, IL-6, iNOS and COX2 [41][42][43]. It has also been reported that selective inhibition of p38 or JNK downregulated TNF-α as well as other inflammatory mediators [44]. These results imply that p38 and JNK MAPK signaling are deeply involved in TNF-α expression.

RAC Decreases Phosphorylated ERK and NF-κB p65 But Not JNK or p38 in LPS-Induced J774A.1 Cells
MAPKs and NF-κB signaling are involved in the TLR family signaling pathway, as part of the downstream signaling cascade [5,6]. Inhibition of these signaling molecules is a good strategy to treat inflammatory diseases. To investigate the effect of RAC on MAPKs and NF-κB signaling molecules, immunoblot analysis was performed. The results showed that RAC treatment downregulated the phosphorylation of ERK and NF-κB p65 ( Figure 3A,B). However, it did not affect the phosphorylation of JNK and p38 ( Figure 3A). These results indicate that RAC selectively regulates signaling molecules, ERK and NF-κB. There are some studies showing that selective inhibition of ERK and NF-κB could not affect the production of TNF-α but decreased other inflammatory mediators, such as IL-1β, IL-6, iNOS and COX2 [41][42][43]. It has also been reported that selective inhibition of p38 or JNK downregulated TNF-α as well as other inflammatory mediators [44]. These results imply that p38 and JNK MAPK signaling are deeply involved in TNF-α expression.

RAC Alleviates Xylene-Induced Mouse Ear Edema
Xylene-induced ear edema is a simple and classic model for acute inflammation studies. It is widely used to evaluate the anti-inflammatory activities of substances [16]. In this study, we assessed the anti-inflammatory effect of RAC on a xylene-induced ear edema model. The administration of PBS, dexamethasone and RAC did not affect intact ear weights ( Figure 4A). The xylene application induced ear weight and differences in ear weight, but dexamethasone and RAC significantly decreased them compared with those of the xylene-only-treated group ( Figure 4B,C).

RAC Alleviates Xylene-Induced Mouse Ear Edema
Xylene-induced ear edema is a simple and classic model for acute inflammation studies. It is widely used to evaluate the anti-inflammatory activities of substances [16]. In this study, we assessed the anti-inflammatory effect of RAC on a xylene-induced ear edema model. The administration of PBS, dexamethasone and RAC did not affect intact ear weights ( Figure 4A). The xylene application induced ear weight and differences in ear weight, but dexamethasone and RAC significantly decreased them compared with those of the xylene-only-treated group ( Figure 4B,C). Acute inflammation is initiated by the recognition of harmful stimuli by immune cells [1,2]. Immune cells, such as macrophages and mastocytes, release proinflammatory mediators like NO, PGE2, histamine, proinflammatory cytokines and chemokines. Histamine, NO and PGE2 cause vasodilation and increased vascular permeability through relaxing smooth muscle [31,45]. Proinflammatory cytokines and chemokines induce cell adhesion molecules, such as ICAM-1 and VCAM-1, recruit immune cells like neutrophils and leukocytes through chemotaxis [46]. These responses cases immune cell infiltration and edema formation. In a histological analysis of the induced ear, xylene application induced severe vasodilation, edema formation, and immune cell Acute inflammation is initiated by the recognition of harmful stimuli by immune cells [1,2]. Immune cells, such as macrophages and mastocytes, release proinflammatory mediators like NO, PGE2, histamine, proinflammatory cytokines and chemokines. Histamine, NO and PGE2 cause vasodilation and increased vascular permeability through relaxing smooth muscle [31,45]. Proinflammatory cytokines and chemokines induce cell adhesion molecules, such as ICAM-1 and VCAM-1, recruit immune cells like neutrophils and leukocytes through chemotaxis [46]. These responses cases immune cell infiltration and edema formation. In a histological analysis of the induced ear, xylene application induced severe vasodilation, edema formation, and immune cell infiltration. The dexamethasone and RAC treatments significantly reduced these factors and skin thickness compared with those of the xylene-only-treated groups ( Figure 4D-G). These results may be due to RAC-downregulated inflammatory mediators, which promote vasodilation and increase vascular permeability.

Materials and Reagents
J774A.1 cells were purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA). The cells were cultured in DMEM (Gibco BRL; Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), 50 U/mL penicillin and 50 mg/mL streptomycin at 37 • C in a 5% CO 2 incubator. Thiazolyl blue tetrazolium bromide, Griess reagent and dexamethasone were obtained from Sigma Aldrich (St. Louis, MO, USA). All antibodies for immunoblot analysis were obtained from Cell Signaling Technology (Danvers, MA, USA).

Isolation of Retrofractamide C
Retrofractamide C was purified from the fruits of Piper longum as previously described [22,23].

MTT Assay
J774A.1 cells were seeded in 96-well plates, treated with 1, 3 and 10 µM of RAC for 24 h and treated with thiazolyl blue tetrazolium bromide for 3 h. After incubation, the supernatant was removed, and the remaining formazan was dissolved in dimethyl sulfoxide (DMSO). The absorbance was measured at 540 nm using a microplate ELISA reader (Molecular Devices, Sunnyvale, CA, USA).

NO Assay
J774A.1 cells were cultured in 96-well plates and treated with 200 ng/mL LPS for 18 h after pretreatment with dexamethasone and RAC for 1 h. Then, the supernatant was collected and treated with Griess reagent. The absorbance was measured at 540 nm using a microplate ELISA reader.

ELISA
J774A.1 cells were seeded in 6-well plates and treated with 200 ng/mL LPS for 18 h after pretreatment with dexamethasone and RAC for 1 h. Then, the supernatant was collected, and the prostaglandin E2 (PGE2) concentration was measured by a mouse PGE2 ELISA kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's instructions. The absorbance was measured at 450 nm using a microplate ELISA reader.

Quantitative Real-Time PCR
J774A.1 cells were seeded in 6-well plates, and cells were pretreated with the indicated concentrations of dexamethasone and RAC and then treated with 200 ng/mL LPS for 12 h. Total RNA was extracted by a PureLink RNA Mini Kit (Invitrogen, San Diego, CA, USA) following the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using a PrimeScript 1st strand cDNA synthesis kit (Takara Bio Inc., Shiga, Japan) and subjected to quantitative real-time PCR. Quantitative real-time PCR was performed by a StepOnePlus Real-Time PCR S machine using a TaqMan probe and TaqMan PCR master mix (Applied Biosystems, Foster City, CA, USA).

Immunoblot Analysis
J774A.1 cells were seeded in 6-well plates and treated with 200 ng/mL LPS for the indicated times after pretreatment with dexamethasone and RAC for 1 h. Total proteins were extracted using cell lysis buffer supplemented with phosphatase and proteinase cocktail (Cell Signaling Technology). The total protein concentration was measured, and equal amounts of protein were subjected to 4-12% SDS-PAGE. Separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and blocked with tris-buffered saline (TBS) containing 5% skim milk. After blocking, the membrane was washed with TBS containing 0.1% Tween-20 (TBST) and incubated with the appropriate primary and secondary antibodies. Finally, the membrane was developed using a West-Queen RTS Western Blot Detection Kit (iNtRON Bio., Seongnam, Korea).

Animals and Induction of a Xylene-Induced Ear Edema Model
Detail procedures were described in a previous study [47]. In brief, six-week-old male ICR mice were purchased from OrientBio (Kwangju, Korea) and randomly divided into 4 groups (n = 9 mice per group): PBS intraperitoneally injected and administered mice (Intact control), PBS intraperitoneally injected and xylene-administered mice (Xylene control), dexamethasone intraperitoneally injected and xylene-administered mice, and RAC orally administered and xylene-administered mice. Before 30 min of topical application of PBS or xylene on the anterior surface of the right ear for 2 h, the mice were administered PBS, 15 mg/kg dexamethasone intraperitoneally and 100 mg/kg RAC orally. After the topical application of 0.03 mL of PBS and xylene on the anterior surface of the right ear, the mice were sacrificed, and changes in ear weight and histopathology were measured. For histological analysis, mouse ears were fixed in 10% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) following the general procedure. The experimental protocols were approved by the Institutional Animal Care and Use Committee of Korea Research Institute of Bioscience and Biotechnology (permission number KRIBB-AEC-17059). All mice were treated according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

Statistical Analysis
The results are presented as the mean ± standard deviation (SD) of three or nine individual experiments. Statistical analysis was performed using Prism 5 software (GraphPad Software, San Diego, CA, USA) for in vitro results and one-way ANOVA followed by Tukey's test for in vivo results.

Conclusions
In this study, we evaluated the anti-inflammatory effect of RAC from Piper longum through in vitro and in vivo experiments. RAC decreased NO and PGE2 production and the protein expression of their synthesis enzymes. The gene expression of the IL-1β and IL-6 proinflammatory cytokines but not TNF-α expression was inhibited by RAC treatment. Immunoblot analysis of MAPKs and