Research Article Piper Cubeba L. Methanol Extract Has Anti-Inflammatory Activity Targeting Src/Syk Via NF-�B Inhibition
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Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2019, Article ID 1548125, 8 pages https://doi.org/10.1155/2019/1548125 Research Article Piper cubeba L. Methanol Extract Has Anti-Inflammatory Activity Targeting Src/Syk via NF-�B Inhibition Nurinanda Prisky Qomaladewi,1 Nur Aziz ,1 Mi-Yeon Kim ,2 and Jae Youl Cho 1 Department of Integrative Biotechnology, Sungkyunkwan University, Suwon , Republic of Korea School of Systems Biomedical Science, Soongsil University, Seoul , Republic of Korea Correspondence should be addressed to Mi-Yeon Kim; [email protected] and Jae Youl Cho; [email protected] Received 19 July 2018; Accepted 23 December 2018; Published 1 January 2019 Academic Editor: Massimo Nabissi Copyright © 2019 Nurinanda Prisky Qomaladewi et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Piper cubeba L. is a plant in the Piperaceae family that is generally found in tropical countries and acts as an antioxidant and anti- infammatory agent. Unfortunately, the molecular mechanism of the anti-infammatory activity has not been fully investigated. In this study, we elucidated the anti-infammatory mechanism by focusing on NF-�B signaling, which is considered a prototypical infammatory signaling pathway in both innate and adaptive immune functions. Cellular activity and the molecular target of Pc-ME were identifed in macrophage RAW264.7 cells and HEK293T cells by assessing NO production, cytokine expression by RT-PCR, luciferase gene reporter assay, and protein regulation in cytoplasm by Western blot upon NF-�B activation. Pc-ME reduced NO production without any cell toxicity; inhibited expression of proinfammatory cytokines such as iNOS and IL-6; downregulated NF-�B activation mediated by both MyD88 and TRIF; and diminished the phosphorylation of I�B�,IKK�/�,Akt,p85,Src,and Syk. Pc-ME inhibited Syk and Src autophosphorylation during overexpression in HEK cells, which confrmed our hypothesis that Syk and Src were signaling targets of Pc-ME. Tese fndings indicate that Piper cubeba L. has anti-infammatory activity by targeting Src/Syk in the NF-�Bpathway. 1. Introduction and activate transcription of infammatory genes such as iNOS, interleukin- (IL-) 6, and other cytokines by activating Infammation is a protective strategy in response to insults transcription factors such as nuclear factor-�B(NF-�B) such as microbial infection, tissue injury, and other noxious [5]. conditions. It causes several classical symptoms, includ- Piper cubeba L. is a Piperaceae plant that is indigenous ing redness, pain, swelling, and heat [1]. In the innate to tropical countries, especially South Borneo and Indonesia. immune system, host cells express various pattern recogni- Important constituents of cubebs are volatile oil, cubebic tion receptors (PRRs) that sense diverse pathogen-associated acid, cubeb resin, and lignans. Some of its lignans, e.g., molecular patterns (PAMPs). Toll-like receptors (TLRs), cubebin and hinokinin, have been reported to have anti- which act as PRRs in mammals, activate signaling path- infammatory and analgesic efects [6]. In vivo,acrude ways that induce expression of several genes involved in hydroalcoholic extract had anti-infammatory and analgesic theimmuneresponse[2,3].UponrecognizingPAMPs, properties [7]. Some reports also found that Piper cubeba L. TLRs dimerize and activate signaling pathways that orig- extract has anti-infammatory activity by attenuating IL-6, inate from conserved cytoplasmic TIR (Toll-Interleukin 1 which is stimulated by lipopolysaccharide (LPS), in THP-1 Receptor) domain-containing adaptors, including MyD88 cells resulting in inhibition of cyclooxygenases (COX-1 and and TRIF [4]. Tese adaptors connect external stimuli to COX-2) and 5-lipoxygenase (5-LOX) [7]. A study also found intracellular signaling molecules such as protein tyrosine that a crude extract of Piper cubeba L. has antioxidant activity, kinases [Syk and Src], serine/threonine kinases [AKT, phos- which could treat skin infammation caused by radicals such phatidylinositide 3 kinases (PI3K), and I�B kinase (IKK)] as ROS and hydrogen peroxide [8]. 2 Evidence-Based Complementary and Alternative Medicine Although previous studies have shown that Piper cubeba Pc-ME and prednisolone were evaluated with an MTT assay L. has anti-infammatory activity, the exact molecular target as previously reported [11]. of this activity has not been investigated. Here we investigated the anti-infammatory activity by using LPS to stimulate TLR4 in vitro. We identifed the molecular targets of this . HPLC Analysis. High-performance liquid chromatogra- extract with various molecular and biochemical approaches. phy (HPLC) analysis was used to confrm the phytochemical characteristics of Pc-ME with standard compounds includ- ing, quercetin, luteolin, and kaempferol [12], as previously 2. Materials and Methods reported [13, 14]. .. Materials. Methanol extract of Piper cubeba (Pc- ME) was purchased from the Plant Extract Bank .. Analysis of mRNA Levels by Reverse Transcription- at the Plant Diversity Research Center (Daejeon, Polymerase Chain Reaction. To determine levels of cytokine Korea; http://extract.kribb.re.kr/extract/f.htm, e-mail: mRNA expression, RAW264.7 cells were treated with Pc- [email protected]). Te cell lines used in these ME (50 and 100 �g/ml) for 30 min and then with LPS experiments were from ATCC (Rockville, MD, USA). (1 �g/ml) for an additional 6 h. Total RNA was extracted with TRIzol Reagent (Gibco RBL) according to the manufac- RAW264.7 cells are a BALB/c-derived murine macrophage ∘ cell line, and HEK293T cells are a human embryonic turer’s instructions, and the isolates were kept at -70 Cuntil kidney cell line. Cell culture products, such as fetal use. Semiquantitative RT-PCR was conducted as previously bovine serum (FBS), penicillin/streptomycin, DMEM, reported [15]. Te primers (Bioneer, Seoul, Korea) used are and RPMI1640, as well as TRIzol were purchased from listed in Table 1. Gibco Products (Grand Island, NY, USA). Phosphate- bufered saline (PBS) was from Capricorn Scientifc .. Preparation of Cell Lysates and Nuclear Fractions for (Ebsdorfergrund, Germany). Polyethylenimine (PEI), Immunoblotting. Afer being treated with Pc-ME for 30 min, Pam3CSK4, lipopolysaccharide (LPS, Escherichia coli 6 RAW264.7 cells (2x10 cells/ml) were treated with LPS for 0111:B4), tetrazole 3-(4,5-dimethylthiazol-2-yl)-2,5- the indicated times. Te cells were washed with cold PBS diphenyltetrazolium bromide (MTT), sodium dodecyl and lysed in lysis bufer (20 mM Tris-HCl, pH 7.4; 2 mM sulfate (SDS), and dimethyl sulfoxide (DMSO) were ethyleneglycotetraacetic acid; 50 mM �-glycerophosphate; 1 obtained from Sigma Chemical Co. (St. Louis, MO, USA). mM sodium orthovanadate; 1 mM dithiothreitol; 1% Triton RT-PCR primers were from Bioneer (Seoul, South Korea). X-100; 10% glycerol; 10 �g/ml aprotinin; 10 �g/ml pepstatin; Luciferase constructs with NF-�B binding promoter sites 1 mM benzamide; and 2 mM PMSF) and centrifugation were used as previously reported [5]. Phospho-specifc and ∘ at12,000rpmfor15minat4C. Te whole cell lysates total antibodies against p50, p65, I�B�,I�B kinase (IKK) ∘ were stored at -70 C until use. To collect nuclear lysates, �/�,Akt(Ser473),p85,Syk,Src,and�-actin were purchased 6 from Cell Signaling Technology (Beverly, MA, USA) and RAW264.7 cells (5x10 cells/ml) were treated with Pc-ME Santa Cruz Biotechnology (Santa Cruz, CA, USA). PP2 and and LPS and washed in cold PBS then washing bufer, which contains 10 mM HEPES (pH 7.8) and KCl, 2 mM piceatannol (Picea) were from Calbiochem (La Jolla, CA, � USA). MgCl2. 0.1 mM EDTA and PMSF, 1 mM DTT, and 2 g/ml leupeptin and aprotinin. Te cells were lysed in washing bufer with 10% Nonidet P-40 then centrifuged at 14000 rpm. .. Cell Culture and Drug Preparation. RAW264.7 and Te pellet (nuclear fraction) was washed in the same bufer, HEK293T were cultured in RPMI1640 and DMEM media, and nuclei were treated with extraction bufer consisting of respectively, supplemented with 1% penicillin/streptomycin, washing bufer with 50 mM KCl, 0.3 M NaCl, 10% glycerol. and 10% (RPMI1640) or 5% (DMEM) FBS. Te cells were ∘ Te extract was centrifuged at 14,000 rpm for 5 min and grown at 37 Cand5%CO2. Stock solution of Pc-ME was the supernatant was collected as the nuclear fraction and ∘ prepared by diluting in concentration of 100 mg/ml using stored at -70 C until use. Soluble cell lysates were analyzed DMSO. by immunoblotting. Proteins were separated on 10% SDS- polyacrylamide gels and transferred by electroblotting onto 6 . Determination of NO Production. RAW264.7 cells (1x10 a polyvinylidenedifuoride (PVDF) membrane. Afer the cells/ml) were preincubated for 18 h [9], treated with Pc- transfer, the membranes were blocked for 60 min in Tris- ME (0-100 �g/ml) or prednisolone as a positive control (0- bufered saline containing 3% BSA and 0.2% Tween 20 at 200 �M) for 30 min, and then treated with LPS (1 �g/ml) or room temperature. Te membranes were incubated at room Pam3CSK4 (10 �g/ml), respectively, for 24 h. Te inhibitory temperature for 60 min with primary antibodies, washed efects of Pc-ME and prednisolone on NO production were three times (10 min each) with the same bufer, and incubated determined with Griess reagent, as previously described [10]. for 60 min with HRP-conjugated secondary antibodies. Te total and phosphorylated levels of p65 (MW: 65), p50 (50), I�B� (40), IKK�/� (85/87), AKT (60), p85 (60-85), Syk 6 .. Cell Viability Assay. RAW264.7 cells (1x10 cells/ml) (72), Src (60), and �-actin (45) were visualized with an ECL were preincubated for 24 h then treated with Pc-ME (0-100 system (Amersham, Little Chalfont, Buckinghamshire, UK) �g/ml) or prednisolone (0-200 �M). Te cytotoxic efects of as previously reported [16]. Evidence-Based Complementary and Alternative Medicine 3 Table 1: Sequences of PCR primers used in this study.