{"paper_id":"59ff7792-33ec-41b2-b929-83b20c28d47b","body_text":"Palmultang (PM) is a traditional herbal medication that has been used since medieval times in East Asia. Currently, PM is usually prescribed as an herbal medicine for the treatment of various symptoms associated with body weakness. Previous studies demonstrated that PM was an effective treatment for endometriosis [ 1 ]. In addition, a recent study revealed that PM has a beneficial effect on reproductive function in female mice [ 2 ]. However, the effects of PM on inflammation and inflammatory mechanisms still remain unknown.\nMacrophages play a key role in the regulation of inflammatory and immune responses [ 3 , 4 ]. Activation of macrophages is induced by LPS stimulation, and activated macrophages secrete inflammatory factors, such as  NO , prostaglandin ( PG ) E 2  and inflammatory cytokines [ 5 , 6 ].  NO  and  PGE 2  are synthesized by inducible nitric oxide synthase ( iNOS ) and cyclooxygenase ( COX ) -2 , respectively, and the expression of  iNOS  is closely related to the induction of  HO-1. HO-1  is a stress-inducible protein that catalyzes the oxidative degradation of heme; two other heme oxygenase isoforms,  HO-2  and  HO-3 , have also been identified [ 7 ]. Enhancing the production of  HO-1  reduces the expression of  iNOS  and the level of free radicals [ 8 ].\nNF-κB  plays an important role in the expression of inflammatory genes. When unstimulated,  NF-κB  is present in the cytoplasm attached to  IκBα; NF-κB  is released through degradation of  IκBα  when induced by LPS [ 9 ]. Activated  NF-κB  can be transferred from the cytoplasm to the nucleus, where it binds to promoters and induces the expression of various inflammatory genes [ 10 , 11 ].  MAPK  signaling pathways play an important role in transmitting inflammatory signals [ 12 ] and comprise extracellular signal-regulated kinase ( ERK ),  p38 , and c-Jun NH 2 -terminal kinase ( JNK ) pathways.  MAPK s are activated by phosphorylation and induce activation of the  NF-κB  pathway and expression of the  iNOS  gene.\nIn the present study, we evaluated the suppressive effect of PM on inflammation induced by LPS in RAW 264.7 macrophages. Further, we researched whether the effects of PM on  NF-κB  and  MAPK  signaling pathways and on induction of  HO-1  explain the anti-inflammatory mechanism of PM.\n\nIn the present study, we demonstrated anti-inflammatory activity of PM in murine macrophages stimulated with LPS. First, we investigated the cytotoxicity of PM in RAW 264.7 macrophages at concentrations of 10–1000 μg/mL. As shown in  Figure 1A , PM did not show cytotoxicity at concentrations up to 1000 μg/mL, indicating that it is not toxic to macrophages. Based on this result, we did experiments using up to 1000 μg/mL concentrations of PM.\nThe overproduction of  NO  is associated with various inflammatory diseases [ 13 , 14 ], so we preferentially investigated the inhibitory effect of PM on the production of  NO  induced by LPS stimulation. As shown in  Figure 1B , the positive control, dexamethasone, which is known to be an anti-inflammatory drug, exerted a strong inhibitory effect on  NO  production. In addition, we discovered that PM dose-dependently repressed  NO  secretion to a statistically significant degree. Notably, PM inhibited  NO  production by more than 70% at a concentration of 500 μg/mL.\nFurther, we examined the inhibitory effect of PM on the production of the pro-inflammatory cytokines tumor necrosis factor ( TNF ) -α , interleukin ( IL ) -6  and  IL-1β . Cytokine expression was analyzed by ELISA and RT-PCR. PM did not inhibit  TNF-α  secretion ( Figure 1C ) and did not suppress the expression of  TNF-α  mRNA ( Figure 1F ). By contrast, PM effectively inhibited both  IL-6  production and mRNA expression in a dose-dependent fashion ( Figure 1D,F ). Likewise, PM strongly suppressed  IL-1β  cytokine and mRNA production at high concentrations ( Figure 1E,F ).\nBecause  COX-2  and  iNOS  are enzymes for  PGE 2  and  NO  synthesis, respectively, we further investigated the inhibitory effects of PM on  COX-2  and  iNOS  expression using Western blots and RT-PCR. As shown in  Figure 2A , PM did not affect expression of  COX-2  at the protein or mRNA level. By contrast, PM showed a strong dose-dependent inhibitory effect on  iNOS  expression that was statistically significant ( Figure 2B ). The inhibitory effect of PM on  iNOS  production was believed to contribute to the suppression of  NO  secretion. These results indicate that PM has inhibitory activity against the production of pro-inflammatory mediators.\nIncreased  HO-1  induction has a direct effect on  iNOS  expression [ 8 ]. Therefore, we investigated whether the inhibitory effect of PM on  iNOS  expression was associated with increased  HO-1  production. We assessed  HO-1  induction in PM-treated macrophages using Western blot and RT-PCR analyses. First, we measured the induction of  HO-1  at 3–24 h after treatment with 1000 μg/mL PM. Protein and mRNA levels of  HO-1  were highest at 6 and 3 h, respectively ( Figure 2C ). Based on the results in  Figure 2C , we investigated  HO-1  protein and mRNA expression at the indicated time points. PM induced  HO-1  expression at the protein and mRNA levels at concentrations of 500 and 1000 μg/mL in a dose-dependent manner ( Figure 2D ). These results suggest that pretreatment with PM inhibits  NO  and  iNOS  production by increasing  HO-1  induction.\nWe demonstrated a repressive effect of PM on secretion of the inflammatory cytokine  IL-6. NF-κB  is a key transcriptional factor associated with the cellular response to stimuli, such as LPS [ 15 – 17 ] and with the production of  NO ,  PGE 2 , inflammatory cytokines, and  iNOS  [ 18 – 20 ]. To investigate whether the inhibitory effect of PM on the expression of inflammatory mediators is associated with activity of the  NF-κB  pathway, we measured the effect of PM on  NF-κB  activation by analyzing translocation of p65 to the nucleus and the phosphorylation of  IκBα . Western blot analysis showed that PM significantly repressed translocation of p65 to the nucleus at a concentration of 100 μg/mL or greater ( Figure 3A ). In addition, the phosphorylation level of  IκBα  was depressed dose-dependently after PM treatment ( Figure 3B ). Thus, PM inhibited the nuclear transcription of p65 by dose-dependently inhibiting  IκBα  degradation induced by LPS stimulation. These findings are consistent with previous studies showing that an  NF-κB  response drives the expression of the  iNOS  and  IL-6  genes [ 21 – 23 ].\nBecause  MAPK s activated by phosphorylation upon LPS stimulation are related to  iNOS  expression and  NF-κB  pathway activation in macrophages [ 24 ], we examined the inhibitory effect of PM on the phosphorylation of  MAPK s. We assessed the phosphorylation levels of  MAPK s, including  ERK 1/2 ,  p38  and  JNK . When RAW 264.7 cells were stimulated with LPS after pretreatment with PM, the levels of phosphorylated  ERK  and  JNK MAPK  were significantly decreased with no change in non-phosphorylated  MAPK  levels ( Figure 4A,C ). By contrast, PM showed only a slight inhibitory effect on  p38  phosphorylation ( Figure 4B ). These results indicate that the inhibitory effect of PM on the phosphorylation of  MAPK s is directly related to inhibition of  NF-κB  activation and reduced production of inflammatory factors in RAW 264.7 cells.\nHPLC-diode array detector (DAD) analysis conditions were successfully established for the separation of peaks in PM extracts. The retention times of eight peaks were as follows: 5-hydroxymethylfurfural (5-HMF), 10.30 min; paeoniflorin, 27.22 min; albiflorin, 30.28 min; ferulic acid, 35.20 min; nodakenin, 36.76 min; decursinol, 43.86 min; glycyrrhizin, 48.69 min; and decursin, 60.93 min.  Figure 5  shows chromatograms of the reference components and of a 60% methanol extract of PM, with detection of eluents at 205 nm (for decursinol), 250 nm (for 5-HMF, albiflorin, ferulic acid, nodakenin, glycyrrhizin, and decursin), 330 nm (for paeoniflorin), with ultraviolet rays (UV) wavelengths selected according to the results of  Figure 6 . These compounds were identified by comparing the retention time and DAD spectra with those of authentic standard compounds. Peak purity checking and identification were conducted using a 190–400 nm UV scan with a DAD.\nCalibration curves were obtained using standard solutions containing 1.25–10,000 μg/mL for 5-HMF, ferulic acid, nodakenin, decursinol, glycyrrhizin, and decursin, 20–20,000 μg/mL for peaoniflorin and albiflorin as marker components. Calibration curve showed good linearity ( r 2  > 0.9990). The limits of detection (LOD) and limits of quantification (LOQ) were 0.16–0.50 μg/mL for 5-HMF, 0.22–0.68 μg/mL for ferulic acid, 0.13–0.40 μg/mL for nodakenin, 0.10–0.29 μg/mL for decursinol, 0.63–0.19 μg/mL for glycyrrhizin and 0.45–0.12 μg/mL for decursin, 48.17–16.05 μg/mL for peaoniflorin, 3.30–10.00 μg/mL for albiflorin ( Table 1 ). The amounts of compounds 1–8 (5-HMF, peaoniflorin, albiflorin, ferulic acid, nodakenin, decursinol, glycyrrhizin, and decursin, respectively) were 11.09, 2.59, 2.30, 3.36, 8.23, 5.11, 0.36, and 1.17 mg/g, respectively. The analytical results for each component identified are summarized in  Table 2 .\nA previous study reported that 5-HMF prevents  TNF-α -induced monocytic cell adhesion to human umbilical vein endothelial cells (HUVECs) by suppression of vascular cell adhesion molecule-1 expression, reactive oxygen species generation and  NF-κB  activation [ 25 ]. Additionally, it was demonstrated that paeoniflorin suppresses  TNF-α -induced chemokine production in human dermal microvascular endothelial cells by blocking  NF-κB  and  ERK  pathways [ 26 ]. Another recent study demonstrated that nodakenin exerts a suppressive effect on LPS-induced inflammatory responses in macrophages by inhibiting TNF receptor-associated factor 6 and  NF-κB  pathways, and it protects mice from lethal endotoxin shock [ 27 ]. A further recent study showed that glycyrrhizin inhibits  NO  and  PGE 2  production in a bimodal fashion [ 28 ]. Another study demonstrated that decursin inhibits induction of inflammatory mediators by blocking  NF-κB  activation in macrophages [ 29 ]. These facts suggest that the anti-inflammatory activity of PM might be related to active components of PM, including 5-HMF, paeoniflorin, nodakenin, glycyrrhizin, and decursin.\n\nProducts related to cell culture (RPMI 1640, fetal bovine serum (FBS) and antibiotics) were purchased from Lonza (Basel, Switzerland). LPS and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO, USA). The Cell-Counting Kit (CCK) was obtained from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Various primary and secondary antibodies for Western blot analysis were purchased from Cell Signaling Technology, Inc. (Boston, MA, USA). Enzyme-linked immunosorbent assay (ELISA) antibody sets for cytokine detection were obtained from eBioscience (San Diego, CA, USA). An RNA extraction kit was purchased from iNtRON (Sungnam, Korea). DNA synthesizing kits and oligonucleotide primers were obtained from Bioneer (Daejeon, Korea). 5-(Hydroxy-methyl)furfural (5-HMF) and ferulic acid were purchased from Sigma (St. Louis, MO, USA). Paeoniflorin and glycyrrhizin were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Decursinol was purchased from Elcom Science (Seoul, Korea), nodakenin from Chem Faces (Wuhan, China), albiflorin from Wako (Osaka, Japan), and decursin from the Ministry of Food and Drug Safety (Osong, Korea). The purity of all representative standards was confirmed by high-performance liquid chromatography (HPLC) to be higher than 97%. HPLC grade solutions, acetonitrile and trifluoroacetic acid were purchased from J. T. Baker Inc. (Philipsburg, NJ, USA). Distilled water (DW) was filtered through a 0.45 μm membrane filter from ADVANTEC (Tokyo, Japan) before analysis.\nPM is composed of eight medicinal herbs listed in  Table 3 . All herbs were purchased from Yeongcheon Herbal Market (Yeongcheon, Korea). All voucher specimens were deposited in an herbal tank, placed in 19,200 mL of DW and then extracted by heating for 3 h at 115 °C and under high pressure (Gyeongseo Extractor Cosmos-600, Inchon, Korea). After extraction, the solution was filtered using standard testing sieves (150 μm) (Retsch, Haan, Germany), freeze-dried and kept in desiccators at 4 °C before use. The acquisition was 591 g and the yield was 30.8%. The freeze-dried extract powder was then dissolved in DW, centrifuged at 14,000 rpm for 10 min and supernatant was filtered (pore size, 0.2 μm) and kept at 4 °C prior to use.\nRAW 264.7 cells were obtained from the Korea Cell Line Bank (Seoul, Korea) and grown in complete RPMI 1640 medium. The cells were incubated in a humidified 5% CO 2  atmosphere at 37 °C. To stimulate the cells, the medium was replaced with fresh RPMI 1640 medium, and LPS (200 ng/mL) was added in the presence or absence of various concentrations of PM (10, 100, 500, and 1000 μg/mL) for the indicated time periods.\nPM was added to the cells, which were incubated for 24 h at 37 °C in 5% CO 2 . CCK solutions were added to each well, and the cells were incubated for an additional 1 h. The optical density was then read at 450 nm using an ELISA reader (Infinite M200, Tecan, Männedorf, Switzerland).\nThe cells were pretreated with PM and stimulated with LPS for 24 h.  NO  production was analyzed by measuring nitrite using Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, 2.5% phosphoric acid) according to a previous study [ 30 ]. Secretion of the inflammatory cytokines  TNF-α ,  IL-6  and  IL-1β  was analyzed using a mouse ELISA antibody set (eBioscience, San Diego, CA, USA). The inhibitory effects of PM were determined at 570 and 450 nm for  NO  and cytokines, respectively, using an ELISA reader.\nThe expression of various proteins was analyzed by Western blot analysis according to standard procedures. Cells were stimulated with LPS with or without PM for the indicated time periods at 37 °C. After incubation, the cells were harvested and resuspended in radio immunoprecipitation assay (RIPA) lysis buffer (Millipore, Bedford, MA, USA) containing protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland) to obtain whole-cell lysates. Cytosolic and nuclear fractions were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, IL, USA) according to the procedure described by the manufacturer. After cell debris was removed by centrifugation, the concentration of protein was determined by Bradford’s method, and equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred onto a nitrocellulose membrane (Millipore, Bedford, MA, USA) and blocked with 3% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBS-T). The membrane was then incubated with each primary antibody at 4 °C overnight, followed by incubation with HRP-conjugated secondary antibodies. The specific proteins were detected using SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA).\nTotal RNA was isolated using an easy-BLUE™ RNA extraction kit (iNtRON, Daejeon, Korea) according to the procedure described by the manufacturer. cDNA was synthesized using AccuPower ®  CycleScript RT PreMix (Bioneer, Daejeon, Korea). The sequences of specific primers used for amplification by polymerase chain reaction are shown in  Table 4 . The following PCR conditions were applied for  TNF-α ,  IL-6 ,  IL-1β ,  COX-2 ,  iNOS ,  HO-1 , and β-actin: 35 cycles of denaturation at 94 °C for 30 s, annealing at the temperature indicated in  Table 4  for 30 s, and extension at 72 °C for 30 s [ 30 – 34 ].\nAn aqua 60% methanol standard stock solution containing compounds 5-HMF, ferulic acid, nodakenin, glycyrrhizin, decursinol, peaoniflorin, albiflorin, and decursin (each 1 mg/mL) were prepared and stored below −4 °C. Working standard solutions were prepared by serial dilution of stock solution with aqua 60% methanol. All calibration curves were obtains from assessement of peak areas of standards in the concentration ranges. A sample of 10 mg PM extract was prepared in 1 mL DW, extracted by ultra-sonication, and filtered through a 0.2 μm syringe membrane filter from Whatman Ltd. (Maidstone, UK) before injection into the HPLC system for analysis. Sample solutions were stored at −4 °C in a refrigerator before analysis.\nAnalytical HPLC data were obtained using an L-2130 pump, L-2200 auto-sampler, L-2300 column oven and L-2455 UV/VIS DAD. The output signal of the detector was recorded using EZChrom Elite software for the HPLC system (Hitachi, Tokyo, Japan). The OptimaPak C 18  analytical HPLC column (4.6 × 250 mm, 5 μm; RS Tech Co., Daejeon, Korea) was used in this study.\nThe mobile phase consisted of water containing (A) 0.1% trifluoroacetic acid and (B) acetonitrile with gradient elution at a flow rate of 1.0 mL/min. The sample injection volume was 20 μL, and the flow rate of the mobile phase was 1.0 mL/min ( Table 5 ). The column temperature was maintained at 40 °C, and the wavelengths of the UV detector were set at 205, 250, and 330 nm.\nThe results are expressed as mean ± SE values. Statistical significance for each treated group compared with the negative control was determined using the Student’s  t  test. Each experiment was repeated at least three times to yield comparable results.  p  values of <0.01 and <0.001 were considered significant.\n\nIn conclusion, PM shows significant inhibitory effects on the secretion of  NO  and expression of  IL-6 ,  IL-1β  and  iNOS  in LPS-stimulated RAW 264.7 cells. These effects are due to inhibition of  NF-κB  activation through suppression of  IκBα  degradation and blockade of  MAPK  phosphorylation. Furthermore, the induction of  HO-1  by PM inhibits inflammatory factor production. These results show that PM could be developed as a new anti-inflammatory agent derived from natural products.","source_license":"CC-BY-4.0","license_restricted":false}