Biochemical and Biophysical Research Communications 463 (2015) 1184e1189

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Ginsenoside Rg3 regulates S-nitrosylation of the NLRP3 inflammasome via suppression of iNOS Sung-Jin Yoon a, c, 1, Jun-Young Park a, c, 1, Song Choi a, Jin-Bong Lee a, c, Haiyoung Jung a, c, Tae-Don Kim a, c, Suk Ran Yoon a, c, Inpyo Choi a, c, Sungbo Shim b, **, Young-Jun Park a, c, * a b c

Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong-gu, Daejeon, Republic of Korea Department of Biomedical Sciences & Neuromarker Resource Bank (NRB), University of Ulsan College of Medicine, Seoul 138-736, Republic of Korea Department of Functional Genomics, University of Science and Technology, Yuseong-gu, Daejeon, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 May 2015 Accepted 11 June 2015 Available online 15 June 2015

Ginsenoside Rg3, a specific biological effector, is well-known as a major bioactive ingredient of Panax ginseng. However, its role in the inflammasome activation process remains unclear. In this report, we demonstrate that ginsenosides 20(R)-Rg3 and 20(S)-Rg3 are capable of suppressing both lethal endotoxic shock and the S-nitrosylation of the NLRP3 inflammasome by inhibiting nitric oxide (NO) production through the regulation of inducible nitric oxide synthase (iNOS) expression. In response to lipopolysaccharide (LPS), the reducing effect of 20(S)-Rg3 and 20(R)-Rg3 on nitric oxide led to an increase in the survival time of mice after lethal endotoxin-induced shock, and excess levels of NO inhibited IL-1b production via the S-nitrosylation of the NLRP3 inflammasome. In addition, ginsenosides 20(R)-Rg3 and 20(S)Rg3 had suppressive effects on the LPS- or UV-irradiation-induced reactive oxygen species (ROS) levels in macrophage and HaCaT cells and thereby prevented apoptosis of spleen cells in mice. Altogether, these results demonstrate that ginsenoside 20(R)-Rg3 and 20(S)-Rg3, a naturally occurring compound, might act as a dual therapeutic regulator for the treatment of inflammatory and oxidative stress-related diseases. © 2015 Elsevier Inc. All rights reserved.

Keywords: Ginsenoside Rg3 NO iNOS S-nitrosylation NLRP3 inflammasome

1. Introduction Ginsenoside Rg3 is one of the most effective steroidal saponins extracted from a traditional medicinal herb, steamed Panax ginseng C.A. Meyer [1]. The major ginsenosides, such as Rb1, Rb2, and Rd, can be converted into a mixture of 20(S)-Rg3 and 20(R)-Rg3 stereoisomers [2]. Rg3 exhibits a wide range of therapeutic and pharmacological properties, including anti-cancer, anti-inflammation, antioxidant, ion channel stereoselectivity, and anti-obesity activities, as well as the ability to affect coronary artery contractions and endothelial cell apoptosis [3e8]. Recently, 20(s)-ginsenoside Rg3 was shown to inhibit autophagy induced by doxorubicin in hepatocellular carcinoma cells and effectively kill HCC cells, in vitro and in vivo [9].

* Corresponding author. Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong-gu, Daejeon, Republic of Korea. ** Corresponding author. E-mail addresses: [email protected] (S. Shim), [email protected] (Y.-J. Park). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.bbrc.2015.06.080 0006-291X/© 2015 Elsevier Inc. All rights reserved.

Nitric oxide (NO) is an important cellular signaling molecule involved in many physiological processes [10]. The production of NO is regulated by a family of nitric oxide synthases (NOSs), and specific NOS subtypes are expressed depending on the tissue type [11]. Recently, the effects of NO and the expression of NOS2 and other NOS isoforms during infectious, autoimmune, chronic inflammatory, malignant, and degenerative diseases in humans and in animal models have been explored to test the therapeutic application of NOS inhibitors or NO donors [12]. The NLR family pyrin domain-containing 3 (NLRP3) inflammasome is a multiprotein complex that activates caspase-1, leading to the processing and secretion of the pro-inflammatory cytokines interleukin-1b (IL1b) and IL-18 [12]. It was recently reported that NO contributes to suppressing the production of IL-1b by S-nitrosylating the NLRP3 inflammasome [13e15] and that Korean red ginseng extracts inhibit NLRP3 and AIM2 inflammasome activation [16]. However, little is known regarding the regulatory mechanism of ginsenoside 20(S)-Rg3 and 20(R)-Rg3 for NO-mediated NLRP3 inflammasome inhibition. In this study, we found that ginsenosides 20(R)-Rg3 and 20(S)-Rg3 negatively regulate LPS-induced endotoxic shock and S-nitrosylation

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2. Materials and methods

Premix Ex Taq (Takara BIO). The mRNA expression levels were calculated using HPRT as the control. The primer sequences were as follows: mouse iNOS, forward 50 -ACA TCG ACC CGT CCA CAG TAT-30 and reverse 50 -CAG AGG GGT AGG CTT GTC TC-3’; HPRT, forward 50 GCC TAA GAT GAG CGC AAG TTG-30 and reverse 50 -TAC TAG GCA GAT GGC CAC AGG-3’.

2.1. Animals

2.5. Detection of S-nitrosylated proteins and biotin pull down assay

All animal-related procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology (KRIBB-IACUC, approval number: KRIBB-AEC-11044), and all procedures were performed in accordance with institutional (National Institutes of Health, USA) guidelines for animal care. All C57BL/6 mice were housed in a pathogen-free animal facility under a standard lightedark cycle with standard rodent chow and water provided ad libitum. Ginsenosides 20(S)-Rg3 and 20(R)-Rg3 with approximately 98% purity were obtained from Hanwool Life Sciences (Daejeon, Korea). Mice were injected intraperitoneally (i.p.) with 10 mg/kg of LPS (Sigma), 10 mg/kg of 20(R)-ginsenoside Rg3 and 10 mg/kg of 20(S)-ginsenoside Rg3 at 10e12 weeks of age. The iNOS inhibitor 1400W (Sigma) was injected intraperitoneally (i.p.) at 20 mg/kg. The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay in spleen was used as previously described [17].

The detection of S-nitrosylated proteins was performed using an S-nitrosylated protein detection assay kit (Cayman), as recommended by the manufacturer. For biotin pull down assay, S-nitrosylated proteins were incubated with NeutrAvidin-agarose resin (Thermo Scientific) for 1 h at room temperature. After 5 washes with washing buffer from the assay kit, the samples were centrifuged at 200  g for 1 min at room temperature [17]. Finally, the detection of S-nitrosylated proteins was performed using western blot. Biotin was detected using the S-nitrosylation detection reagent-tagged HRP provided with the assay kit.

of the NLRP3 inflammasome by inhibiting NO production through the suppression of iNOS expression. In addition, the possibility of these compounds downregulating ROS production was examined using a macrophage and HaCaT human keratinocyte cell line.

2.2. Cell culture Raw 264.7 cells (murine macrophage) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), penicillin G (100 IU/ml), and streptomycin (100 mg/ ml). HaCaT cells (human keratinocyte) were grown in DMEM media supplemented with 10% FBS and antibiotics. Mouse peritoneal macrophages were harvested 4 days after the mice were injected with 3% thioglycollate (Sigma). Peritoneal macrophages were plated in 6-well plates at 2.0  106 cells per well. After incubation for 2 h at 37  C, the wells were washed three times to remove nonadherent cells. Finally, the media was changed to RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin G (100 IU/ml), and streptomycin (100 mg/ml). Bone marrow cells were isolated from C57BL/6 mice and cultured, as described previously [17]. For the preparation of bone marrow-derived macrophages (BMDM), bone marrow was differentiated in 20% (vol/vol) L929 cell conditioned medium for 6 days. BMDM were plated in 6-well plates at 2.0  106 cells per well. 2.3. Western blot analysis Cells were incubated in RIPA buffer containing a protease inhibitor cocktail, and tissues were homogenized using a Precellys®24 homogenizer (Bertin Technologies). The protein content of the cells and tissues was determined using a BCA protein assay kit (Pierce, USA) and adjusted to 20 mg for analysis. For western blot analysis, antibodies specific for pAkt and Akt were purchased from Cell Signaling Technology. Antibodies for iNOS, ASC1, caspase-1 and b-actin were purchase from Santa Cruz Biotechnology, and NLRP3 antibody was purchased from Adipogen. 2.4. RNA isolation and quantitative real-time PCR Total RNA was isolated using an RNA isolation kit (Qiagen), as recommended by the manufacturer. Total RNA (1 mg) was reversetranscribed to generate first-strand cDNA and analyzed using realtime PCR with a Dice TP 800 Thermal Cycler (Takara BIO) with SYBR

2.6. Nitrite determination and enzyme-linked immunosorbent assay (ELISA) The concentration of nitrite in the serum, liver, and spleen samples was determined using a NO (total) detection kit (Enzo Life Sciences). Liver and spleen tissue was harvested, immediately flash frozen, and homogenized in a buffer containing 1 mM protease inhibitor cocktail. NO detection in culture media was performed using Griess reagent (Sigma) at 540 nm. Serum and macrophage culture media were analyzed for IL-1b content using DuoSet antibody pairs (R&D Systems) according to the manufacturer's instructions. 2.7. Measurement of intracellular reactive oxygen species (ROS) level The intracellular reactive oxygen species were detected using the fluorescent probe CM-H2DCFDA (Molecular Probes, USA). Briefly, cells were harvested and then stained with 10 mM CMH2DCFDA in the dark for 30 min at 37  C. In preparation for UV-B irradiation, HaCaT cells were washed with PBS; the cells were then irradiated with UV-B (0.1 J/cm2) without a plastic lid. UV-B irradiated cells were stained with 10 mM CM-H2DCFDA in the dark for 30 min at 37  C. Cells were washed once with phosphatebuffered saline (PBS) in pre-warmed growth medium for an additional 10 min at 37  C, and fluorescence was analyzed using a flow cytometer and confocal microscopy. The images were captured using an LSM510 confocal microscope (Carl Zeiss). 2.8. Caspase-3/7 activity The detection of caspase-3/7 activity was performed using Caspase-Glo® 3/7 Assay (Promega), as recommended by the manufacturer. Cells were plated in 96-well plates at 5.0  104 cells per well and then incubated with LPS after treatment with 20(R)ginsenoside Rg3 and 20(S)-ginsenoside Rg3 for 16 h. The culture media was removed by washing the wells 3 times with phosphate buffered saline (PBS), then Caspase-Glo® 3/7 reagent was added to the wells, and luminescence was recorded for 3 h on a MicroLumat Plus LB96v luminometer (Berthold Technologies). 2.9. Statistical analysis Quantitative data are presented as the mean ± standard deviation (SD) from representative experiments (n ¼ 3). Statistical

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analyses were carried out using one-tailed Student's t-tests. A value of p < 0.05 and p < 0.01 was considered statistically significant.

and 20(S)-Rg3 in peritoneal macrophages and BMDM after LPS stimulation.

3. Results

3.2. Effect of ginsenoside 20(R)-Rg3 and 20(S)-Rg3 on reactive oxygen species (ROS)

3.1. Effect of ginsenoside 20(R)-Rg3 and 20(S)-Rg3 on iNOS and nitric oxide (NO) Two optical isomers of ginsenoside 20(R)-Rg3 and 20(S)-Rg3 have been found in ginseng products. They are epimers of each other depending on the position of the hydroxyl (OH) group on carbon-20 (Fig. 1). To examine the effect of ginsenoside 20(R)-Rg3 and 20(S)-Rg3 on inflammation activation, we evaluated NO production levels in Raw264.7 cells with various dosages of 20(R)-Rg3 and 20(S)-Rg3 (0, 5, 10 and 20 mM) for 16 h after LPS stimulation (Fig. 1B). Additionally, LPS-induced NO production in mouse peritoneal macrophages and bone marrow-derived macrophages (BMDM) was reduced by 20(R)-Rg3 and 20(S)-Rg3 (Fig. 1C and D). Next, using western blot analysis, we found that treatment with 20(R)-Rg3 and 20(S)-Rg3 after LPS stimulation suppressed iNOS protein levels in peritoneal macrophages and BMDM (Fig. 1E and F). In addition, iNOS mRNA was decreased by treatment with 20(R)Rg3 and 20(S)-Rg3 in peritoneal macrophages and BMDM after LPS stimulation (Fig. 1G and H). Overall, these data suggest that 20(R)Rg3 and 20(S)-Rg3 decrease the production of NO and the expression levels of both iNOS protein and mRNA in LPS-stimulated macrophage cells. Overall, these data suggest that the production of NO and expression of iNOS were negatively regulated by 20(R)-Rg3

Reactive oxygen species (ROS), such as oxygen-derived superoxide anions, hydrogen peroxide, and hydroxyl radicals, are initiators of toxic oxidative reactions. To assess whether 20(R)-Rg3 and 20(S)-Rg3 can suppress the production of LPS-induced ROS, we assessed ROS levels in peritoneal macrophages using FACS and confocal microscopy analysis. As shown in Fig. 2A and B, ROS levels were decreased by treatment with 20(R)-Rg3 and 20(S)-Rg3. Additionally, 20(R)-Rg3 and 20(S)-Rg3 effectively reduced the levels of ROS in HaCaT cells irradiated with 0.1 J/cm2 UV-b (Fig. 2C). The cysteine aspartic acid-specific protease (caspase) family plays a key effector role in apoptosis in mammalian cells. To assess whether 20(R)-Rg3 and 20(S)-Rg3 could inhibit apoptosis, we tested the caspase-3/7 activity in peritoneal macrophages. 20(R)Rg3 and 20(S)-Rg3 could inhibit caspase-3/7 activity in these cells (Fig. 2D). These results indicate that 20(R)-Rg3 and 20(S)-Rg3 are able to diminish the ROS levels and prevent ROS-induced apoptosis. 3.3. Effects of ginsenoside 20(R)-Rg3 and 20(S)-Rg3 on the Snitrosylation of the NLRP3 inflammasome and Akt NO is one of the signal regulators of protein function via protein S-nitrosylation. The suppression of both NO production and iNOS

Fig. 1. Effects of 20(R)-Rg3 and 20(S)-Rg3 on NO production and iNOS gene expression in LPS-stimulated macrophages. (A) The structure of 20(R)-ginsenoside Rg3 and 20(S)ginsenoside Rg3. (B) Raw 264.7 cells were cultured with 500 ng/ml LPS after pre-incubation with 20(R)-Rg3 and 20(S)-Rg3 (5, 10 and 20 mM). NO levels in the culture medium were measured after 16 h using Griess reagent. (C) Peritoneal macrophages were cultured with 100 ng/ml LPS after pre-incubation with 20(R)-Rg3 and 20(S)-Rg3 (20 mM), and NO levels in the culture medium were measured after 16 h using Griess reagent. (D) Bone marrow-derived macrophages (BMDM) were cultured with 500 ng/ml LPS after pre-incubation with 20(R)-Rg3 and 20(S)-Rg3 (20 mM), and NO levels in the culture medium were measured after 16 h using Griess reagent. (E) iNOS protein levels in peritoneal macrophages detected by western blotting after incubation of 20(R)-Rg3 and 20(S)-Rg3 (5, 10 and 20 mM) for 16 h. (F) Protein levels of iNOS in BMDM detected by western blotting after incubation with 20(R)-Rg3 and 20(S)-Rg3 (20 mM) for 16 h (G) iNOS mRNA levels in peritoneal macrophages were evaluated by real-time PCR analysis after incubation with LPS alone or with 20(R)Rg3 and 20(S)-Rg3 (10 and 20 mM) for 16 h. Hprt was used as a housekeeping gene control. (H) mRNA levels of iNOS in BMDM were evaluated by real-time PCR analysis after incubation with LPS alone or with 20(R)-Rg3 and 20(S)-Rg3 (10 and 20 mM) for 16 h. Hprt was used as a housekeeping gene control. (*p < 0.05, **p < 0.01 compared with LPS alone).

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Fig. 2. Effects of 20(R)-Rg3 and 20(S)-Rg3 on ROS production in LPS-stimulated peritoneal macrophages. (A) ROS levels were measured by FACS analysis using CM-H2DCFDA (10 mM) incubate with LPS 100 ng/ml at 30 min after pre-treated with 20(R)-Rg3 and 20(S)-Rg3 (20 mM). (B) ROS levels in peritoneal macrophages were determined by performing immunofluorescence using CM-H2DCFDA (20 mM). (C) HaCaT cells were irradiated with 0.1J/cm2 UV-b after incubation with 20(R)-Rg3 and 20(S)-Rg3 (20 mM). (D) Peritoneal macrophages were treated with LPS (100 ng/ml) after pre-incubation with 20(R)-Rg3 and 20(S)-Rg3 (20 mM), and then cell lysates were harvested at 24 h. The caspase-3 activity assay was measured by luminescence signal using Luminometer. (*p < 0.05, **p < 0.01 compared with LPS alone).

expression by 20(R)-Rg3 and 20(S)-Rg3 might be involved in NOmediated NLRP3 inhibition. To evaluate this hypothesis, we analyzed the S-nitrosylation of NLRP3 inflammasome proteins, including NLRP3, ASC, and caspase-1, in peritoneal macrophages. The S-nitrosylation levels of NLRP3 and caspase-1 were increased when cells were treated with LPS for long exposure times (12 and 16 h), but these responses to LPS were significantly decreased by treatment with 20(R)-Rg3 and 20(S)-Rg3 (Fig. 3A). In addition, we assessed the S-nitrosylation of the NLRP3 inflammasome in BMDM and found that the S-nitrosylation in response to LPS of NLRP3 and caspase-1 was dramatically decreased in incubated BMDM with 20(R)-Rg3 and 20(S)-Rg3 treatment (Fig. 3B). Considering these results together, we assessed the level of IL-1b in peritoneal macrophages and BMDM in response to treatment with 20(R)-Rg3 and 20(S)-Rg3 compared with LPS alone. The production of IL-1b as a result of the S-nitrosylation of inflammasome components in response to LPS was increased by treatment with 20(R)-Rg3 and 20(S)-Rg3 (Fig. 3C). Next, to determine whether NO would affect the activity of Akt, we tested the S-nitrosylation of Akt in peritoneal macrophages. We found that the S-nitrosylation levels of Akt in response to LPS were decreased by treatment with 20(R)-Rg3 and 20(S)-Rg3; in addition, Akt phosphorylation was increased by 20(R)-Rg3 and 20(S)-Rg3 treatment (Fig. 3D). These results indicate that excessive NO production by long-term LPS-stimulated peritoneal macrophages and BMDM might inhibit IL-1b processing, which results from the activation of the inflammasome and Akt phosphorylation, and this processing is recovered by the inhibition of S-nitrosylation via the prevention of excessive NO production by treatment with 20(R)-Rg3 and 20(S)-Rg3. Overall, these results suggest that 20(R)-Rg3 and 20(S)-Rg3 could inhibit the S-nitrosylation of the NLRP3 inflammasome and Akt by preventing excessive NO production. 3.4. Effect of ginsenoside 20(R)-Rg3 and 20(S)-Rg3 on LPS-induced endotoxic shock To determine whether 20(R)-Rg3 and 20(S)-Rg3 would affect susceptibility to endotoxic shock in vivo, we injected mice with LPS.

Following the injection of LPS (10 mg/kg body weight) and ginsenoside Rg3 (20(R)-Rg3 10 mg/kg and 20(S)-Rg3 10 mg/kg body weight), the survival of mice was assessed every 5 h. Almost all of the control mice, which received an LPS only injection, died within 40 h, whereas the mice injected with 20(R)-Rg3 and 20(S)-Rg3 were more likely to survive until 80 h and 120 h (Fig. 4A). Furthermore, apoptosis was analyzed in the spleen, using TUNEL assays 18 h after LPS injection, and a small number of apoptotic cells were detected in the spleen from 20(R)-Rg3 and 20(S)-Rg3-injected mice compared with that observed in mice injected with LPS alone (Fig. 4B). We also measured the expression of iNOS in the liver after LPS injection, and similar to the results observed for the spleen, iNOS expression and NO production in the liver were markedly decreased in 20(R)-Rg3 and 20(S)-Rg3-injected mice compared with LPS only-injected mice (Fig. 4C and D). Next, to confirm LPSinduced NO in vivo, we measured NO in the serum, liver and spleen of mice. LPS-induced NO was significantly decreased in organs and serum by treatment with 20(R)-Rg3 and 20(S)-Rg3 (Fig. 4E). Overall, these data suggest that 20(R)-Rg3 and 20(S)-Rg3 play an essential role in suppressing sensitivity to lethal endotoxininduced shock by regulating the production of NO in vivo. 4. Discussion Ginsenoside Rg3, one of the natural ginsenosides in Panax ginseng, has specific biological characteristics, such as antioxidant effects [3], ion channel stereoselectivity [5], coronary artery contraction effects [6], peroxisome proliferator-activated receptor (PPAR) pathway regulation [8] and endothelial cell apoptosis [7]. In this study, we demonstrated that Rg3 suppressed iNOS, NO and ROS levels in macrophage and HaCaT cells. NO is an important cellular signaling molecule involved in many physiological processes, as well as the killing of intracellular pathogens [10]. It has been reported that NO has a relationship with NO-mediated NLRP3 inflammasome inhibition via the S-nitrosylation of the NLRP3 inflammasome [14e17]. However, little is known regarding the regulatory mechanism of ginsenoside 20(S)-Rg3 and 20(R)-Rg3 for NO-mediated NLRP3 inflammasome inhibition. As shown in Fig. 1

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Fig. 3. 20(R)-Rg3 and 20(S)-Rg3 reduced NO production and increased the activation of the NLRP3 inflammasome and Akt via S-nitrosylation. (A) The total level of S-nitrosylation of inflammasome components was determined by immunoblot analysis. Peritoneal macrophages were treated with LPS alone or were pre-incubated with 20(R)-Rg3 and 20(S)-Rg3 (20 mM) for 16 h Nlrp3 and caspase-1 were detected by biotin pull down assay. Bottom (lysate), immunoblot analysis of total lysate fractions. (B) The total level of S-nitrosylation of inflammasome components was determined by immunoblot analysis. BMDM were treated with LPS alone or pre-incubated with 20(R)-Rg3 and 20(S)-Rg3 (20 mM) for 16 h Nlrp3 and caspase-1 were detected by biotin pull down assay. Bottom (lysate), immunoblot analysis of total lysate fractions. (C) peritoneal macrophages (Left) and BMDM (Right) were pre-stimulated with 20(R)-Rg3 and 20(S)-Rg3 (20 mM) for 1 h and then primed with LPS 100 ng/ml and 500 ng/ml for 16 h. IL-1b secretion was measured in the culture supernatants by ELISA. (D) The total level of S-nitrosylation of Akt was determined by immunoblot analysis. Peritoneal macrophages were treated with LPS alone or were pre-incubated with 20(R)-Rg3 and 20(S)-Rg3 (10 and 20 mM) for 16 h (*p < 0.05 compared with LPS alone).

and 3, ginsenoside 20(S)-Rg3 and 20(R)-Rg3 could reduce NO generation and iNOS expression and prevent the S-nitrosylation of the NLRP3 inflammasome in the long-term LPS-stimulated

macrophages and BMDM. In previous reports, Akt enhanced the survival of cells by blocking the function of proapoptotic proteins. However, Akt is inactivated by S-nitrosylation via NO in vitro and in

Fig. 4. 20(R)-Ginsenoside Rg3 and 20(S)-ginsenoside Rg3 protect more mice against lethal endotoxin-induced shock by regulating the production of NO in vivo. (A) 20(R)-Rg3 and 20(S)-Rg3 (10 mg/kg body weight) was injected i.p into C57BL/6 mice (n ¼ 15), and then LPS (10 mg/kg body weight) was injected i.p. (B) TUNEL assays were performed on sections of spleens from mice at 18 h post-LPS injection. Quantitation of the TUNEL staining is shown. We counted apoptotic cells within 6 randomly selected fields. (C) Tissue lysate samples (20 mg) were harvest liver and spleen using homogenizer, and then protein levels of iNOS were detected by Western blotting. (D) Real-time PCR was performed with tissue cDNA samples to measure iNOS mRNA levels in liver and spleen. Hprt was used as a housekeeping gene control. (E) After 20(R)-Rg3 and 20(S)-Rg3 (10 mg/kg) injection in C57BL/6 mice (n ¼ 5 per group) treated with LPS (10 mg/kg), serum liver and spleen NO levels were determined. (*p < 0.05, **p < 0.01 compared with LPS alone).

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intact cells. In this study, we found that ginsenoside 20(S)-Rg3 and 20(R)-Rg3 can prevent the suppression of Akt phosphorylation by S-nitrosylation-mediated inactivation [18,19]. ROS, such as oxygen-derived superoxide anions, hydrogen peroxide, and hydroxyl radicals, are initiators of toxic oxidative reactions [20,21]. In addition, inflammasome activation might be controlled by mitochondrial ROS generation. It was previously reported that the thioredoxin-interacting protein (TXNIP) binds with NLRP3, and then this complex activates the NLRP3 inflammasome [22]. ROS induce the dissociation of TXNIP from thioredoxin in a reactive oxygen species (ROS)-sensitive manner; dissociated TXNIP/ thioredoxin complex might be linked with anti-inflammatory effects and the S-nitrosylation of the NLRP3 inflammasome [17]. In Fig. 2, we showed that ginsenoside 20(S)-Rg3 and 20(R)-Rg3 negatively regulates the production of ROS, suggesting that this compound might inhibit NLRP3 inflammasome activation via ROS suppression. NO might synergize with superoxide to induce cell death via peroxynitrite production. High NO concentrations (>500 nM) induce cell death, mainly either by oxidative/nitrosative stressinduced apoptosis or by energy depletion-induced necrosis [23,24]. As shown in Fig. 4, the mortality of mice and apoptosis of spleen cells were recovered by ginsenoside 20(S)-Rg3 and 20(R)Rg3. These findings support that ginsenoside 20(S)-Rg3 and 20(R)Rg3 has a suppressive effect on the generation of NO and ROS in both mortality of mice and cell death. Overall, we demonstrated that ginsenosides 20(R)-Rg3 and 20(S)-Rg3 play a critical role in the control of both lethal endotoxic shock and the S-nitrosylation of the NLRP3 inflammasome by inhibiting NO production through the regulation of iNOS expression. In addition, excess levels of NO inhibit IL-1b production via the S-nitrosylation of the NLRP3 inflammasome. We also observed that ROS levels were decreased by treatment with ginsenoside 20(R)Rg3 and 20(S)-Rg3 in macrophage and HaCaT cells and demonstrated that diminished ROS by ginsenoside Rg3 prevented the apoptosis of spleen cells in mice. Subsequently, the survival ratio of mice was significantly increased by treatment with ginsenoside 20(S)-Rg3 and 20(R)-Rg3. In conclusion, this work demonstrates that ginsenoside 20(R)-Rg3 and 20(S)-Rg3, a naturally occurring compound, might act as a dual therapeutic regulator for the treatment of inflammatory and oxidative stress-related diseases. Acknowledgments This work was supported by grants from the KRIBB Research Initiative Program, Republic of Korea (KGM4931511), and by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIP) (NRF2013M3A9B6046567). We thank Dr. Ik-Soo Kim (Hanwool Life Sciences) for providing ginsenoside 20(R)-Rg3 and 20(S)-Rg3. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.06.080.

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Ginsenoside Rg3 regulates S-nitrosylation of the NLRP3 inflammasome via suppression of iNOS.

Ginsenoside Rg3, a specific biological effector, is well-known as a major bioactive ingredient of Panax ginseng. However, its role in the inflammasome...
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