Immunology Letters 158 (2014) 143–150
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Korean red ginseng extracts inhibit NLRP3 and AIM2 inﬂammasome activation Jeeyoung Kim a,1 , Huijeong Ahn a,1 , Byung-Cheol Han b , Seung-Ho Lee b , Young-Wook Cho c , Cheon Ho Kim a , Eui-Ju Hong e , Beum-Soo An d , Eui-Bae Jeung e , Geun-Shik Lee a,∗ a
College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon 200-701, Republic of Korea Korea Ginseng Corp., Daejeon 305-805, Republic of Korea c Korean Basic Science Institute, Chuncheon 200-701, Republic of Korea d Department of Biomaterial Science, College of Natural Resources and Life Science, Pusan National University, Gyeongsangnam-do 627-706, Republic of Korea e Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungcheongbuk-do 361-763, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 12 September 2013 Received in revised form 19 December 2013 Accepted 20 December 2013 Available online 10 January 2014 Keywords: Red ginseng extracts Cytokine Inﬂammasome Interleukin-1 Macrophages AIM2
a b s t r a c t Korean red ginseng extract (RGE) is one of the most popular natural herbs modulating the immune system. Although the effects of RGE on immunity have been reported, its effects on inﬂammasomes, multi-protein complexes that activate caspase-1 to induce maturation of interleukin (IL)-1␤, have not been studied yet. In this study, we elucidated the effect of RGE on inﬂammasome activation using mouse and human macrophages. In our results, RGE inhibited IL-1␤ maturation resulting from NLRP3 inﬂammasome activation in both in vitro and in vivo models. In addition, RGE strongly attenuated IL-1␤ secretion as well as pathogen clearance via pyroptotic cell death by macrophages through inhibition of AIM2 inﬂammasome activation. Ginsenosides Rg1 and Rh3 were suggested as inhibitors of the inﬂammasome activation. Thus, we demonstrated that RGE inhibits both NLRP3 and AIM2 inﬂammasome activation, with predominant involvement of the AIM2 inﬂammasome. Crown Copyright © 2014 Published by Elsevier B.V. All rights reserved.
1. Introduction Korean red ginseng (Panax Ginseng C.A Meyer) is one of the most well studied traditional medicinal herbs. Ginseng can be separated into red ginseng and white ginseng according to commercially use. Naturally dried ginseng is known as white ginseng, whereas red ginseng is made by steaming and drying fresh ginseng root. Korean red ginseng contains various active components, including saponins such as ginsenosides and non-saponins such as polysaccharides, peptides, fatty acids, and mineral oils . Ginsenosides are the major active component of ginseng and has vasorelaxation, anti-oxidation, and anti-cancer effects . Ginsenosides exhibit anti-inﬂammatory properties by inhibiting NF-B activation in association with reduced secretion and/or mRNA expression of proinﬂammatory mediators . Ginsenosides are also amphiphilic in
∗ Corresponding author. Tel.: +82 33 250 8683; fax: +82 33 244 2367. E-mail address: [email protected]
(G.-S. Lee). 1 These authors contributed equally to this work.
nature and have the ability to intercalate into the plasma membrane . As ginsenosides are signaling molecules that are similar to steroid hormones, they can alter membrane ﬂuidity and function as well as elicit cellular responses when moving through the plasma membrane [5,6]. Red ginseng has been studied for many years as an immune-modulating substance, and previous studies have determined it to be an adjuvant or remedy for the treatment of infectious diseases as well as many other metabolic disorders . Interleukin (IL)-1␤ is an important mediator of inﬂammation that increases synthesis of cytokines, febrile disease progression, gene regulation of endometrial cells, chemotaxis, leukocyte adhesion, ﬁbroblast activity, and heart rate . IL-1␤ is generated by inﬂammasomes , which are cytoplasmic multi-protein complexes that mediate the maturation of IL-1␤ by activating caspase-1 . The primary activity of inﬂammasomes is the activation of caspase-1. In detail, the caspase-1 catalyzes pro-IL-1␤ produced by PRRs signaling into mature IL-1␤ . Most cytokines are activated as they are simultaneously generated and secreted, but IL-1␤ is ﬁrst generated as a pro-form (pro-IL-1␤) before being converted into
0165-2478/$ – see front matter. Crown Copyright © 2014 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.imlet.2013.12.017
J. Kim et al. / Immunology Letters 158 (2014) 143–150
its ﬁnal active form by inﬂammasome activation. Inﬂammasomes have been found to regulate other important aspects of inﬂammation and tissue repair such as pyroptosis, a form of cell death [11,12]. Mutation of just one inﬂammasome-associated gene has been shown to induce auto-inﬂammatory disease . Several inﬂammasomes have been identiﬁed to date [10,14], including NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inﬂammasome, which recognizes various danger signals, AIM2 (absent in melanoma 2) inﬂammasome, which recognizes dsDNA, and NLRC4 (NLR Family, CARD domain-containing 4) inﬂammasome, which recognizes ﬂagellin. Although the anti-inﬂammatory effects of Korean red ginseng extracts (RGE) have been widely reported, the effects of RGE on inﬂammasome activation have not been identiﬁed. In this study, we investigated the effects of RGE on IL-1␤ regulation and inﬂammasome activation in bone marrow-derived macrophages (BMDMs). We observed that RGE inhibited both NLRP3 and AIM2 inﬂammasome activation. 2. Materials and methods 2.1. Cell culturing Unless otherwise indicated, all materials for cell culture were obtained from PAA (GE Healthcare Bio-Sciences Co., NJ, USA). Bone marrow-derived macrophages (BMDMs) were obtained by differentiating bone marrow progenitors from tibia and femur bones using L929 cell-conditioned medium (LCCM) as a source of granulocyte/macrophage colony-stimulating factor . Progenitors were cultured in RPMI 1640 (LM 011-01, Welgene, Seoul, Republic of Korea) supplemented with 10% fetal bovine serum (FBS), 30% LCCM, 100 U/ml of penicillin, and 100 g/ml of streptomycin. Cells were seeded in non-tissue culture-treated Petri dishes (SPL life science Co., Gyeonggi-do, Republic of Korea) and incubated at 37 ◦ C in a 5% CO2 atmosphere for 7 days. THP-1 cells were obtained from Korean Cell Line Bank (KCLB No. 40202; Seoul, Republic of Korea) and maintained in RPMI 1640 medium containing 10% FBS, 100 U/ml of penicillin, and 100 g/ml of streptomycin at 37 ◦ C in a 5% CO2 atmosphere. THP-1 cells were plated in 12-well plates (1 × 106 cells per well) and differentiated to macrophage-like cells with phorbol 12-myristate acetate (100 nM, PMA; Invivogen, CA, USA) for 72 h. For cytotoxicity assay, BMDMs (10,000 cells/well) were plated in a 96-well plate (SPL Life Science Co.) and allowed to be attached for 3 h. The BMDMs were treated with the indicated dosage of RGE for 1 h and the cytotoxicity was measured by Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., MD, USA) as manufacture’s protocol. 2.2. Korean red ginseng extract (RGE) Korean RGE was manufactured from the roots of a 6-yearold fresh Panax ginseng and provided by Korea Ginseng Corp. (Daejeon, Republic of Korea). The provided RGE was composed 33.75% of water, 5.5% of crude ash, 12.96% of crude protein, and 47.79% of carbohydrate. In addition, RGE contained Rb1 (9.29 mg/g), Rb2 (3.18 mg/g), Rc (3.60 mg/g), Rd (1.00 mg/g), Re (2.45 mg/g), Rf (1.06 mg/g), Rg1 (2.00 mg/g), Rg2 (0.90 mg/g), Rg3 (1.90 mg/g), Rh1 (0.65 mg/g) and other minor ginsenosides. 2.3. Inﬂammasome activation and inhibition The PMA treated THP-1 and BMDMs (1.0 × 106 cells per well) were plated on 12-well plates (SPL life science Co.) and primed with 10 g/ml of lipopolysaccharide (LPS; Sigma–Aldrich Co., MO, USA) in RPMI 1640 containing 10% FBS and antibiotics for 3 h. After LPS priming, BMDMs or THP-1 cells were subjected to the following
activation steps for 1 or 3 h. For NLRP3 inﬂammasome activation, the medium was replaced with RPMI 1640 supplemented with ATP (2 mM; InvivoGen, CA, USA) for 1 h, nigericin (NG, 40 M; Tocris Bioscience, Bristol, UK) for 1 h, calcium pyrophosphate dihydrate (CPPD; 200 g/ml; InvivoGen) for 1 h, CaCl2 (1 mM; Biosesang, Seoul, Republic of Korea) for 1 h, aluminum potassium sulfate (Alum; 200 g/ml; Daejeung Chemicals & Materials Co., Gyeonggido, Republic of Korea) for 3 h. For NLRC4 inﬂammasome activation, the medium was exchanged with Salmonella typhimurium (OD600: 1.2, 0.5%) and collected after 1 h. For AIM2 inﬂammasome activation, LPS-primed BMDMs were supplemented with 2 g/ml of dsDNA containing 4 l/ml of Lipofectamine 2000 (Invitrogen, CA, USA) for 1 h or Listeria monocytogenes (OD600: 0.3, 10%) for 3 h. In addition, E. coli (DH5␣, OD600: 1.0, 1%) was used as a negative control. RGE, Quillaja saponin (Junsei Chemical Co., Tokyo, Japan), ginsenoside Rh1 (ChromaDex® Co., CA, USA), or ginsenoside Rg3 (ChromaDex® Co.) were co-treated with the above activators. Due to the cytotoxicity of saponin, Rh1, and Rg3 on BMDMs (data not shown), the optimized concentrations as indicated in the ﬁgure were adopted in the current experiment. Cellular supernatant (Sup), lysate (Lys), and cross-linked pellets (Pellet) with suberic acid bis (Sigma–Aldrich Co.) were collected for further analysis.
2.4. Western blot analysis Sup, Lys, and Pellet samples were separated by SDS-PAGE (10% or 16%) and blotted onto a polyvinylidene diﬂuoride membrane (Pall Co., NY, USA). Immunoblots were probed overnight at 4 ◦ C with anti-mouse IL-1␤ antibody (AF-401-NA, R&D Systems, MN, USA), anti-human IL-1␤ antibody (AF-201-NA, R&D systems), anticaspase-1 p20 antibody (06-503, EMD Millipore Co, MA, USA), anti-caspase-1 antibody (sc-622, Santa Cruz Biotechnology), antiAsc antibody (sc-22514, Santa Cruz Biotechnology, CA, USA), c-Myc antibody (SC-40, Santa Cruz Biotechnology), or anti-actin antibody (sc-1615, Santa Cruz Biotechnology). The membranes were further probed with HRP-conjugated 2nd anti-sera (sc-2020 or sc-2004, Santa Cruz Biotechnology) and visualized by Power-Opti ECLTM solution (BioNote Co., Gyeonggi-do, Republic of Korea) and a cooled CCD camera System (AE-9150, EZ-Capture II, ATTO Technology, Tokyo, Japan).
2.5. IL-1ˇ detection using ELISA To quantitate the secreted IL-1␤, the cell culture supernatants of BMDMs were measured by mouse IL-1beta/IL-1F2 Quantikine ELISA Kit (R&D Systems). The ELISA plates were readout using a microplate reader (Bio-Red, CA, USA).
2.6. Animals Male C57BL/6 mice (8-week-old) were purchased from Narabio Co. (Seoul, Republic of Korea). All mice were maintained under a 12 h light/dark cycle at 24 ◦ C. Animals were provided standard sterile food and water ad libitum, after which they were allowed to adjust to the environment for 1 week. For LPS-induced endotoxin shock, mice were fed 2% of RGE (n = 11), 10% of RGE (n = 10) or water (n = 21) ad libitum for 1 week before LPS (20 mg/kg) intraperitoneal injection. Animals were observed every 8 h for 3 days. Animal care and all experimental procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Kangwon National University (ACUCC; approval no. KIACUC-12-0009).
J. Kim et al. / Immunology Letters 158 (2014) 143–150
2.7. Transfection efﬁciency assay Transfection was carried out using LipofectamineTM 2000 (Invitrogen) like dsDNA-mediated AIM2 inﬂammasome activation. 293 T cells (3 × 105 cells) were plated on 6-well plates (SPL Lifesciences) 1 day before transfection. Plasmid (2 g) expressing both c-Myc and red ﬂuorescence protein (RFP) was mixed with serum-free DMEM containing LipofectamineTM 2000 (Invitrogen), after which the mixture was added to wells. After incubation for 1 h, cell culture media were replaced with DMEM containing 10% FBS with antibiotics for 17 h. RFP signals were observed with a ﬂuorescence microscope (Nikon, Tokyo, Japan) and cellular lysates were subjected to Western blot assay.
inﬂammasome activation (Fig. 1D). The inhibitory properties of RGE on NLRP3 inﬂammasome were further conﬁrmed in human macrophages (THP-1 cells, Fig. 1F). The inhibitory effect of RGE on NLRP3 inﬂammasome activation was further conﬁrmed using an LPS-induced endotoxin shock model. According to the literature, susceptibility to LPS-induced endotoxin shock is reduced in NLRP3 gene-deleted mice compared to intact mice . As shown in Fig. 1G, 2 or 10% of RGE orally treated mice showed higher survival rates than control mice 1 week prior to LPS injection. Thus, RGE attenuated the severity of NLRP3 inﬂammasome-mediated disease symptoms in animals, which is similar to our in vitro result in which RGE inhibited IL-1␤ maturation in BMDMs. Hence, RGE directly inhibited NLRP3 inﬂammasome activation and could be applied as an NLRP3 inﬂammasome inhibitor.
2.8. Bacterial growth 3.2. Korean red ginseng inhibits AIM2 inﬂammasome activation S. typhimurium and L. monocytogenes were obtained from the Korean Culture Center of Microorganisms (KCCM; Seoul, Republic of Korea). Salmonella and Listeria were grown on Luria-Bertani (LB, Laboratories Conda, Madrid, Spain) and Brain Heart Infusion (BHI, Laboratories Conda) agar plates for 18 h at 37 ◦ C. A single colony was transferred to LB or BHI broth, after which the culture was incubated for 18 h with shaking at 37 ◦ C. Listeria host invasion assay was performed as previously described . Brieﬂy, BMDMs (1 × 106 cells per well or 5 × 104 cells per well) were plated on 96-well plates and inoculated with Listeria (2 × 107 colony-forming units [cfu] or 1 × 105 cfu) in DMEM containing 10% FBS for 1 or 12 h. The plate was then washed with Gentamycin (50 g/ml; Komipharm International Co., Ltd., Gyeonggi-do, Republic of Korea) containing PBS to eliminate extracellular Listeria, followed by plating on a BHI plate to calculate cfu. 2.9. Statistical analyses Statistical analyses were performed using a t-test for the two groups or one-way ANOVA for multiple groups, and survival analysis for lethality test using GraphPad Prism (GraphPad Software, San Diego, CA). 3. Results 3.1. Korean red ginseng inhibits NLRP3 inﬂammasome activation To elucidate the effect of red ginseng extracts (RGE) on inﬂammasome activation, we treated LPS-primed BMDMs with various dosages of RGE alone or RGE with ATP, a well-characterized NLRP3 inﬂammasome activator, and measured IL-1␤ (p17, active form) secretion by immuno blotting (Fig. 1A) and ELISA assays (Fig. 1B). As shown in Fig. 1A (left side), mature IL-1␤ (p17) was not detected in the cell supernatant (Sup) of the RGE alone treated cells suggesting that RGE did not activate inﬂammasomes. While we treated LPS-primed BMDMs with RGE in the presence of ATP to estimate the inhibitory effect of RGE on IL-1␤ secretion resulting from NLRP3 inﬂammasome activation. RGE dose-dependently inhibited ATP-mediated IL-1␤ secretion. In addition, RGE effectively interrupted the formation of Asc pyroptosomes, which are a typical indicator of inﬂammasome activation. As expected, the RGE treatment did not induce any cytotoxicity in the BMDMs (Fig. 1C). Further, we treated LPS-primed BMDMs with other NLRP3 inﬂammasome activators, including nigericin (NG; Fig. 1D) and aluminum potassium sulfate (Alum; Fig. 1E) in the presence of RGE to conﬁrm inhibition of NLRP3 inﬂammasome activation. Whereas production of mature IL-1␤ was observed in macrophages treated with NG or Alum alone, RGE dose-dependently inhibited NLRP3 inﬂammasome activation. In addition, RGE attenuated the secretion of the active form of caspase-1 (p20) by NG-mediated NLRP3
We further tested the inhibitory effect of RGE on NLRC4 and AIM2 inﬂammasome activation. The NLRC4 inﬂammasome is activated by intracellular bacteria containing ﬂagellin, whereas the AIM2 inﬂammasome activation is triggered by intracellular dsDNA derived from the host or pathogens . As shown in Fig. 2A, we inoculated S. typhimurium (Salmonella) for NLRC4 inﬂammasome activation or transfected dsDNA into LPS-primed BMDMs for AIM2 inﬂammasome activation, after which IL-1␤ maturation and Asc pyroptosome formation were analyzed. Although RGE did not block IL-1␤ maturation or Asc pyrotosome formation induced by Salmonella, it strongly interrupted dsDNA-mediated IL-1␤ secretion and Asc pyrotosome formation. Interestingly, less than 600 ppm of RGE completely attenuated IL-1␤ (Fig. 2B) and caspase-1 (Fig. 2C) secretions. Thus, RGE could be an effective AIM2 inﬂammasome inhibitor. We further explored the mechanism of AIM2 inﬂammasome induction. To conﬁrm whether or not RGE inhibited dsDNA transfection, a plasmid expressing Myc and RFP proteins was transfected into 293 T cells in the presence of various dosages of RGE (Fig. 2D). Although the highest dosage of RGE did reduce transfection efﬁciency, all other RGE concentrations that inhibited AIM2 inﬂammasome activation had no effect on Myc and RFP expressions. In addition, we inoculated L. monocytogenes (Listeria), which triggers both AIM2 and NLRP3 inﬂammasome activation with predominant involvement of the AIM2 inﬂammasome , into LPS-primed BMDMs in the presence of RGE (Fig. 2E). RGE signiﬁcantly inhibited Listeria-induced IL-1␤ secretion, although the degree of inhibition was less potent than that of dsDNA-mediated IL-1␤ secretion. In the same vein as the transfection efﬁciency test (Fig. 2D), we inoculated Listeria into BMDMs and measured intracellular bacteria number (cfu) after 1 or 12 h of inoculation. As shown in Fig. 2F, the invasion rate of Listeria into BMDMs was not altered by the presence of RGE, indicating that RGE directly inhibited intracellular inﬂammasome activation. In addition, the number of Listeria after 12 h of inoculation signiﬁcantly increased in the presence of RGE, implying that RGE interrupted clearance of Listeria via pyroptotic cell death  (Fig. 2G). The inhibitory effects of RGE on AIM2 inﬂammasome were further conﬁrmed in human macrophages (Fig. 2H). Thus, RGE was a potent inhibitor on AIM2 inﬂammasome activation. 3.3. Ginsenosides inhibit inﬂammasome activation We next attempted to identify which material of red ginseng is responsible for the inhibition of inﬂammasome activation. We ﬁrstly focused on saponins (Quillaja saponins) and ginsenosides (Rh1 and Rg3; Fig. 3A), which are the major pharmacological constituents of ginseng . We treated LPS-primed BMDMs with saponin, Rh1, or Rg3 along with the inﬂammasome activators NG or dsDNA. As shown in Fig. 3B, IL-1␤ secretion
J. Kim et al. / Immunology Letters 158 (2014) 143–150
Fig. 1. RGE inhibits NLRP3 inﬂammasome activation. BMDMs (1 × 106 cells per well) were primed by LPS (10 g/ml) in RPMI medium containing 10% FBS for 3 h, after which the medium was replaced with RPMI medium containing the indicated percentages of RGE with ATP (2 mM, A and B) or NG (40 M, D) for 1 h or Alum (200 g/ml, E) for 3 h. Cell culture supernatants (Sup), cell lysates (Lys), and cross-linked pellets (Pellet) from whole-cell lysates were analyzed by immunoblotting as indicated. Secreted IL-1␤ was quantitated by an ELISA based assay kit and presented with bar graphs (B, D, and E). (C) The cytotoxicity of RGE was measured after applying the indicated dosage of RGE on BMDMs for 1 h like to the inﬂammasome activating step. Triton ×-100 (0.01%, Triton) suggested by the manufacturer presents complete cell death. Triton treated group sets as 0% of the survival rate and non-treated group sets as 100%. (F) THP-1 (1 × 106 cells per well) were differentiated by PMA (100 nM) for 3 days and then primed by LPS (10 g/ml) for 3 h, after which the medium was replaced with RPMI medium containing the indicated percentages of RGE with ATP (2 mM) or NG (40 M) for 1 h or Alum (200 g/ml) for 3 h. Supernatants and cell lysates were analyzed by immunoblotting as indicated. All immunoblot data shown are representative of three or more independent experiments. All data for bar graphs represent the mean ± s.e.m. of three independent experiments, each performed in triplicate. (G) Mice were treated with 2 or 10% of RGE or water ad libitum for 1 week before LPS (20 mg/kg) intraperitoneal injection. Survival rates were observed for 3 days. P < 0.05, RGE (2 and 10%) treated mice vs. Water supplied mice.
was unaltered by saponin. However, the highest concentration of Rh1 moderately inhibited NG or dsDNA-induced IL-1␤ secretion (Fig. 3C). In addition, Rg3 ginsenoside attenuated inﬂammasome activator-mediated IL-1␤ maturation (Fig. 3D). Interestingly, Rg3 was the most potent inhibitor of dsDNAmediated AIM2 inﬂammasome activation. Thus the ginsenosides derived from RGE interrupted NLRP3 and AIM2 inﬂammasome activation. 4. Discussion The anti-inﬂammatory properties of RGE have been previously studied , although there is currently no study on the
effect of RGE on inﬂammasome activation mediating IL-1␤ maturation. In this study, we observed that RGE inhibited two well characterized inﬂammasomes, NLRP3 and AIM2. That is, RGE inhibited IL-1␤ secretion from NLRP3 activator-treated BMDMs and/or THP-1, and it ameliorated LPS-induced lethality. In addition, RGE strongly attenuated IL-1␤ secretion and pyroptosome formation in dsDNA-transfected macrophages. Attenuation of pyrototic cell death induced intracellular Listeria growth. As expected, ginsenosides Rh1 and Rg3 were shown to be key inhibitors of inﬂammasome activation, whereas saponins derived from quillaja did not alter inﬂammasome activation. Thus, ginsenosides of RGE act as inhibitors of both NLRP3 and AIM2 inﬂammasome activation.
J. Kim et al. / Immunology Letters 158 (2014) 143–150
Fig. 2. Effects of RGE on dsDNA- or Listeria-mediated AIM2 inﬂammasome activation. (A) LPS-primed BMDMs were co-treated with NLRC4 or AIM2 inﬂammasome activators, Salmonella (OD600: 1.2, 0.5%), or dsDNA (2 g/ml) in Lipofectamine (4 l/ml) as well as the indicated percentages of RGE. Cell culture supernatants (Sup), cell lysates (Lys), and cross-linked pellets (Pellet) from whole-cell lysates were analyzed by immunoblotting as indicated. The secreted IL-1␤ was quantitated with an ELISA-based assay and presented as bar graphs. Data represent the mean ± s.e.m. of three independent experiments. (B) The inhibitory property of RGE on dsDNA-mediated IL-1␤ secretion was progressively observed in the present of serial dilution of RGE. (C) Caspase-1 maturation and secretion was detected by Western blotting assay. (D) Plasmid expressing Myc and red ﬂuorescence protein (RFP) was introduced into 293 T cells with Lipofectamine 2000 in the presence of RGE. Red ﬂuorescence signals were observed with ﬂuorescence microscope. Bar indicated 100 m. Myc band intensity was measured to elucidate the effect of RGE on transfection efﬁciency. Myc expression without RGE was set as 100%, and the non-transfected sample was set as 0%. Data represent the mean ± s.e.m. of three independent experiments. (E) LPS-primed BMDMs were inoculated with the indicated bacteria with or without RGE and measured for IL-1␤ secretion. BMDMs were inoculated with Listeria for 1 h (F) or 12 h (G), after which the number of intracellular bacteria was calculated. cfu of the Listeria-inoculated group was set as 100%, and data represent the mean ± s.e.m. of four independent experiments, each performed in triplicate. (H) Human macrophages (THP-1) were primed by LPS and then treated Salmonella, dsDNA, or Listeria in the present of RGE. Supernatants and cell lysates were analyzed by immunoblotting as indicated.
J. Kim et al. / Immunology Letters 158 (2014) 143–150
Fig. 3. Effects of ginsenosides on NLRP3 or AIM2 inﬂammasome activation. (A) The chemical structures of ginsenosides, Rh1 and Rg3, were adopted from PubChem (http://pubchem.ncbi.nlm.nih.gov). LPS-primed BMDMs were treated with NG (40 M) or dsDNA (2 g/ml) in Lipofectamine (4 l/ml) in the presence of saponins (Quillaja saponins, (B) ginsenoside Rh1, (C) or ginsenoside Rg3 (D). Cell culture supernatants (Sup) and cell lysates (Lys) were analyzed by immunoblotting as indicated.
Although several inﬂammasomes have been identiﬁed , their molecular mechanisms of activation have been poorly characterized, except for the NLRP3 inﬂammasome. Brieﬂy, inﬂammasome activation might be controlled by intracellular potassium efﬂux, mitochondrial reactive oxygen species (ROS) generation, and
lysosomal rupture [18,21]. In addition, intracellular Ca2+ and cAMP have been suggested as alternative molecular effectors in the regulation of NLRP3 inﬂammasome activation . Among these, we have focused on mitochondrial ROS [23–25] and intracellular Ca2+ [22,26,27] as mediators of the inhibitory effect of RGE on NLRP3
J. Kim et al. / Immunology Letters 158 (2014) 143–150
inﬂammasome, as they have been frequently reported as targets of RGE. That is, ginseng extract has been shown to reverse elevation of ROS generation [28–30] and intracellular Ca2+ [31–33] in various cell types. Based on this evidence, we conclude that RGE inhibits NLRP3 inﬂammasome activation. To elucidate the inhibitory effect of RGE on inﬂammasome activation in an animal model, we performed an LPS lethality test (Fig. 1G). As expected, RGE-fed mice showed higher survival rates than control mice. Similar data have previously been reported . In a study by Ahn et al., pretreatment with ginsan, a polysaccharide extracted from P. ginseng, protected mice from lethality induced by septic challenge, and ginsan-treated mice displayed lower levels of serum pro-inﬂammatory cytokines such as IL-1␤ . In addition, ginsan reduced the expression of toll-like receptor (TLR)-2, -4, -9, and MyD88, thereby protecting mice from sepsis . Based on this report, we speculate that RGE inhibits both IL-1␤ transcription and maturation via inﬂammasome activation. RGE not only inhibited NLRP3 inﬂammasome activation but also strongly attenuated dsDNA-induced IL-1␤ secretion. Cytoplasmic dsDNA sensed by AIM2 interacts with Asc, resulting in the formation of the Asc pyroptosome for induction of pyroptotic cell death in cells containing caspase-1 [18,35]. Pyroptosis is a critical pro-inﬂammatory cell death mechanism involving caspase-1 activity in which inﬂammasomes mediate pathogen clearance . To conﬁrm the inhibitory effect of RGE on Asc pyroptosome formation, we used L. monocytogenes, which displays predominantly AIM2 inﬂammasome activation over NLRP3 inﬂammasome activation . The invasion rate of Listeria was unaffected by RGE treatment, whereas the growth rate of Listeria was signiﬁcantly elevated. This result suggests that RGE inhibits both AIM2 and NLRP3 inﬂammasome activation and interrupts clearance of Listeria. Ginsenosides can be divided into protpanaxadiol (PPD) and protophanaxatriol (PPT) ginsenosides according to their basic structure . PPD type ginsenosides include ginsenosides-Rb1, Rb2, -Rc, -Rd, -Rg3, and -Rh, whereas PPT type ginsenosides include ginsenosides-Re, -Rf, -Rg1, -Rg2 and -Rh1 . In the current study, we selected two ginsenosides, Rg3 or Rh1, and saponins (Quillaja saponins) and determined their effects on inﬂammasome activation. Rh1, which possesses anti-allergic and anti-inﬂammatory activities, has been recently reported to ameliorate obesity and myocardial injury in the rodent models [36–38]. While Rg3 has been progressive studied as an anti-carcinogenic agent and it also presents several biological activities including anti-oxidant, antiangiogenesis, and anti-inﬂammatory [39,40]. Rg3 is found mainly in red ginseng and is derived from the hydrolysis of saponins by heat processing . Although both Rh1 and Rg3 have showed anti-inﬂammatory activities, the effect of these on inﬂammasome activation has not been studied. As shown in Fig. 3, saponins did not inhibit NLRP3 or AIM2 inﬂammasome activation however Rh1 and Rg3 attenuated NG- or dsDNA-induced IL-1␤ secretion. Especially, Rg3 seemed to more potently inhibit dsDNA-induced IL-1␤ secretion. Other studies have proposed that various metabolic diseases are associated with inﬂammasome activation . Indeed, treatments targeting IL-1␤ using an IL-1 receptor antagonist or IL-1␤ monoclonal antibody have been suggested to cure arthritis, type 2 diabetes, arteriosclerosis, or Alzheimer’s disease [42–44]. However, these targeted treatments impose large ﬁnancial burdens as well as unwanted side effects on patients. Hence, selective inﬂammasome inhibitors are needed such as glyburide, a type 2 diabetes drug, and the NF-B inhibitors Parthenolide and Bay11-7082, which were reported as selective inhibitors of NLRP3 inﬂammasome activation [45,46]. In the current study, we suggest RGE as an inhibitor of inﬂammasome activation or an alternative source for IL-1␤targeted treatments.
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