International Immunopharmacology 24 (2015) 182–190
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Suppression of nuclear factor-kappa B and mitogen-activated protein kinase signalling pathways by goshonoside-F5 extracted from Rubi Fructus Jian-Ming He a,b,1, Shi-Cai Chen c,1, Run-Ping Li d, Liang-Xi Yuan e, Jun-Min Bao e,⁎, Mei-Li Guo a,⁎⁎ a
Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai 200433, China School of Pharmacy, Fudan University, Shanghai 201203, China c Department of Otorhinolaryngology, Changhai Hospital, Shanghai 200433, China d Department of Diving Medicine, Faculty of Navy Medicine, Second Military Medical University, Shanghai 200433, China e Department of Vascular Surgery, Changhai Hospital, Second Military Medical University, Shanghai 200433, China b
a r t i c l e
i n f o
Article history: Received 19 September 2014 Received in revised form 3 November 2014 Accepted 3 December 2014 Available online 15 December 2014 Keywords: Rubi Fructus Ent-labdane diterpene glucoside Macrophage Anti-inflammatory NF-κB MAPK
a b s t r a c t Rubi Fructus, a traditional Chinese medicine, was considered as an anti-inflammatory agent in folk medicine. In the present study, we investigated the signalling pathways involved in the anti-inflammatory effects of goshonoside-F5 (GF5), isolated from Rubi Fructus, in peritoneal macrophages and examined its therapeutic effect in a mouse endotoxic shock model. GF5 decreased NO and PGE2 production in LPS-stimulated macrophages (IC50 = 3.84 and 3.16 μM). This effect involved the suppression of NOS-2 and COX-2 gene expression at the transcriptional level. Examination of the effects of GF5 on NF-κB signalling demonstrated that it inhibits the phosphorylation of IκB-α and IκB-β, blocking their degradation and the nuclear translocation of the NF-κB p65 subunit. Moreover, inhibition of MAPK signalling was also observed, and phosphorylation of p38 and JNK was suppressed in the presence of GF5. Inflammatory cytokines, including IL-6 and TNF-α, were down-regulated by this compound after activation with LPS (IC50 = 17.04 and 4.09 μM). Additionally, GF5 (30 and 90 mg/kg, i.p.) significantly reduced the circulating cytokine levels (IL-6 and TNF-α) and increased survival in a mouse model of endotoxemia. These results show that GF5 significantly inhibits the pro-inflammatory response induced by LPS, both in vitro and in vivo. Our results provide a strong pharmacological basis for further understanding the potential therapeutic role of GF5 in inflammatory disease and shed new light on the bioactivity of ent-labdane diterpene glucoside. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Inflammation is a host response by an organism to foreign pathogens or tissue injury to eliminate harmful stimuli, including infection and noxious stimuli, as well as to promote healing and repair processes in the damaged tissue [1]. Macrophages play a key role in the acute immune response by recognising and phagocytosing pathogens, releasing cytokines and chemokines, processing antigens and repairing tissue damage [2]. Upon the activation of Toll Like Receptor 4 (TLR4) by LPS in macrophages, MAPKs become phosphorylated and also phosphorylate the IκB kinase (IKK) complex, eventually activating the NF-κB and MAPK pathways, resulting in the production of NO, PGE2 and various cytokines, such as TNF-α, IL-1β and IL-6. NO and PGE2 are generated in
⁎ Corresponding author. Tel.: +86 21 81861566. ⁎⁎ Corresponding author. Tel./fax: +86 21 81871302. E-mail addresses: [email protected] (S.-C. Chen), [email protected] (J.-M. Bao), [email protected] (M.-L. Guo). 1 Co-first author.
http://dx.doi.org/10.1016/j.intimp.2014.12.007 1567-5769/© 2014 Elsevier B.V. All rights reserved.
macrophages by the inducible isoforms of cyclooxygenase (COX-2) and NO synthase (NOS-2), respectively. These pro-inflammatory cytokines and signalling molecules play important roles in inflammatory diseases and pathological conditions [3–7]. Rubus chingii Hu, a member of the Rosaceae family, is extensively grown in both north and south China. Rubi Fructus, the dried unripe fruit of R. chingii, has been used as a food and tonic for thousands of years. In traditional Chinese medicine, this fruit is proposed to give tone to the kidney and liver [8]. Moreover, it is considered as an antiinflammatory agent in folk medicine to treat diseases such as urinary tract infections, nephritis, chronic hepatitis and enteritis. In modern pharmacological experiments, the anti-oxidant [9], hepatoprotective [10], antifatigue [11] and immunomodulatory [12] properties of Rubi Fructus have been reported, and it has additional effects on the hypothalamus–pituitary–gonad axis [13]. However, the anti-inflammatory compounds derived from Rubi Fructus have rarely been reported. Our previous phytochemistry study of Rubi Fructus resulted in the isolation of various compounds classified as flavonoids (isoquercetin, kaempferol-3-O-β-Drutinoside, tiliroside and kaempferol), benzaldehydes and organic
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Fig. 1. Chemical structure and HPLC chromatogram of GF5.
acids (vanillic aldehyde, 4-Hydroxy-3-methoxybenzoic acid and pHydroxybenzaldehyde), steroids (β-sitosterol, Stigmast-5-en-3-ol, Oleate), and ent-labdane diterpenes (goshonoside-G, goshonoside-F2, goshonoside-F4 and goshonoside-F5) [14,15]. We reported the potential anti-inflammatory activity of the novel compound goshonoside-G [15]. However, goshonoside-F5 (GF5) is also a major compound (Fig. 1) in Rubi Fructus. The present study was designed to investigate the antiinflammatory effects of GF5 in LPS-activated macrophages and in mice undergoing endotoxic shock. We further examined whether GF5 could reduce inflammatory responses through the suppression of the NF-κB Table 1 NMR data (δ in ppm) of GF5 (C5D5N). Position
GF5 δH (J in Hz)
δC
1.20 (m), 1.77 (m) 1.64 (m) 3.75 (1H, t, J = 8 Hz)
5.32 (1H, t, J = 7 Hz) 4.31 (1H, s), 4.82 (1H, s) 1.68 (3H, s) 4.52 (1H, s), 4.82 (1H, s) 3.25, 3.81 (1H, d, J = 9.5) 0.73 (3H, s) 0.68 (3H, s)
38.4 28.2 73.3 44.2 48.0 25.3 39.2 150.1 57.1 40.5 23.2 39.7 142.9 121.8 66.6 16.8 107.2 74.4 13.0 15.8
15-O-Glc 1′ 2′ 3′ 4′ 5′ 6′
4.29 (1H, d, J = 8 Hz) 3.22 (m) 3.26 (m) 3.30 (m) 3.35 (m) 3.64 (m), 3.84 (m)
103.0 75.2 78.1 72.0 78.5 63.1
18-O-Glc 1″ 2″ 3″ 4″ 5″ 6″
4.22 (1H, d, J = 8 Hz) 3.18 (m) 3.27 (m) 3.26 (m) 3.35 (m) 3.64 (m), 3.84 (m)
105.0 75.3 78.3 72.2 78.6 63.2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1.85 (m) 1.35 (m), 1.70 (m) 2.07 (m), 2.33 (m) 1.82 (m) 1.46 (m), 1.62 (m) 1.88 (m), 2.15 (m)
and MAPK signalling pathways. The following is our first report of this study. 2. Materials and methods 2.1. Materials ESI-MS was measured on a Varian MAT-212 mass spectrometer. Silica gel (200–300 mesh, Qingdao Haiyang Chemical Co. Ltd., Qingdao, China). The 1H NMR and 13C NMR spectra were recorded on a Bruker DMX-400 NMR spectrometer with tetramethylsilane (TMS) as an internal standard. HPLC assays were conducted using an Agilent 1100 HPLC (Agilent Technologies, Palo Alto, CA, USA). Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FBS) and penicillin–streptomycin solution were obtained from Gibco (Carlsbad, CA, USA). Lipopolysaccharide (LPS) (Escherichia coli strain 0111:B4) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for phospho-IκB-α, phospho-IκB-β, IκB-α, IκB-β, p-65, phospho-p38, p38, phospho-JNK and COX-2 were obtained from EPITOMICS (San Diego, CA, USA). Antibodies against phospho-p42–p44 ERK, p42–p44 ERK and COX-2 were purchased from Cell Signalling Technology (Danvers, MA, USA). Anti-JNK, anti-β-actin and anti-NOS-2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Goat anti-rabbit IgG-HRP secondary antibodies and Cy3 goat anti-rabbit antibodies were from KPL (Gaithersburg, MD, USA). Enzyme linked immunosorbent assay (ELISA) kits for PGE2, TNF-α and IL-6 were from R&D Systems (Minneapolis, MN, USA). Male C57BL/6Slac mice (6–8 weeks old) were obtained from SLRC Laboratory Animal Co., Ltd. (Shanghai, China). All procedures were conducted in accordance with the University guidelines and approved by the Ethical Committee for Animal Care and the Use of Laboratory Animals at Second Military Medical University. C57BL/6Slac mice free of pathogens were bred in our animal facility and supplied with food and water ad libitum and exposed to a 12 h light–dark cycle. 2.2. Preparation of GF5 and its purity 2.2.1. Isolation of GF5 from Rubi Fructus Dry Rubi Fructus (1000 g) was extracted three times using 70% ethanol as a solvent. The solvent was evaporated under reduced pressure to give an ethanol extract, which was further subjected to chromatography using a column packed with macroporous resin D101 (Chemical Plant of NanKai University, Tianjin, China) and eluted with water and 30%, 60%, and 95% aqueous ethanol. The 60% ethanol extract (130 g) was further fractionated by MCI-gel CHP20P (Mitsubishi Chemical Co., Tokyo) column chromatography using a gradient of aqueous ethanol
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2.2.2. High-performance liquid chromatography (HPLC) The purity of GF5 was determined by high performance liquid chromatography (HPLC) using an Agilent Series 1100 liquid chromatograph coupled with evaporative light scattering detection (ELSD). Chromatography was performed through an Agilent Zorbax-C18 column (5 μm, 250 mm × 4.6 mm, i.d.), which was controlled at 35 °C using acetonitrile (A) and H2O (B) as the mobile phase under a gradient programme with a flow rate of 1.0 ml/min: 0–5 min, 75–69% A; 5–12 min, 69–65% A. The ELSD was programmed with the following settings: evaporator temperature, 40 °C; air pressure, 3.5 bar. The purity of goshonoside-F5 was determined to be 98.7% by normalisation of the peak areas detected by HPLC (Fig. 1). 2.3. Preparation of peritoneal macrophages
Fig. 2. Effects of GF5 on cell viability. Peritoneal macrophages were incubated with different concentrations of GF5 (0.1, 1,10, 20, 50 and 100 μM) in the presence or absence of 100 ng/ml LPS for 24 h. Cell viability was determined by the CCK-8 assay. The results shown are the means ± S.D. of three independent experiments.
(20, 30, 40, 50, 60, 80 and 100%) to give a 60% ethanol fraction. The 60% ethanol fraction was then separated using Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden) column chromatography and eluted with EtOH–H2O (2:5 v/v) to yield the investigated compound in present study (white powder, 487 mg), which was stained purple with ethanolic vanillin–sulphuric acid. The structure of this compound was further identified using characteristic and spectral methods. The ESI mass spectrum in positive mode showed quasimolecular ion peaks at m/z 669 [M + Na]+, indicating a molecular mass (M 664) corresponding to the formula C32H54O13. The structure of this compound was determined as goshonoside-F5 (GF5), and spectroscopic data was shown in Table 1, which agrees with spectra in the literature [16].
Peritoneal macrophages were isolated from the C57BL/6Slac mice essentially as previously described [17], with slight modification. Briefly, the mice were intraperitoneally (i.p.) injected with sterilised culture broth (1 mL). On day 3 following the injection, the peritoneal exudate cells were isolated by washing the peritoneal cavity with ice-cold PBS. The cells were seeded at 1 × 106/cm2 in DMEM supplemented with 10% FBS and 2% penicillin–streptomycin antibiotic at 37 °C in a 5% CO2 incubator. Two hours later, the culture medium was replaced to remove the non-adherent cells. The adherent cells were incubated at 37 °C in a humidified 5% CO2 atmosphere overnight and used as peritoneal macrophages for the subsequent procedures. 2.4. Assay for cell viability Macrophages were plated at a density of 105 cells/well in 96-well plates. To determine the appropriate non-toxic treatment concentrations, cells were incubated in the presence of different concentrations of GF5 for 24 h. The viable cell content was measured using colorimetric cell counting kit 8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan). The
Fig. 3. Cytokine release is inhibited by GF5. (A) Macrophages were treated with GF5 (0.1, 1, 10, and 50 μM) or a control treatment for 30 min followed by 4 h of stimulation with 100 ng/ml LPS. mRNA levels of IL-6 and TNF-α were determined by real-time PCR. (B) Macrophages were treated with GF5 (0.1, 1, 10, 20, 50 and 100 μM) or a control treatment for 30 min followed by 24 h of stimulation with 100 ng/ml LPS. The production of IL-6 and TNF-α was determined in the supernatant by ELISA. The results shown are the means ± S.D. of three independent experiments carried out in triplicate. *P b 0.05, **P b 0.01, ***P b 0.001 vs. LPS.
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assays were performed in triplicate, and the results are expressed as the percent reduction in cell viability compared with untreated control cultures for at least three independent experiments.
2.5. Assay of inflammatory mediator and cytokine release 2.5.1. Determination of NO biosynthesis The nitric oxide (NO) content of the culture supernatant was measured using the Griess Reagent (Beyotime Institute of Biotechnology, Jiangsu, China) by determining the concentration of NaNO2 produced by NO metabolism [18].
2.5.2. Assay of PGE2 release The levels of PGE2 in the culture supernatant were quantified using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.
2.5.3. Cytokine assay Cytokine (IL-6 and TNF-α) production by cultured macrophages was determined using an enzyme-linked immunosorbent assay (ELISA) kit from R&D systems according to the manufacturer's instructions.
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2.6. Western blot analysis The treated cells were washed twice with ice-cold PBS. Total cellular protein was extracted using the Protein Extraction Reagent (Goodbio, Wuhan, China). Protein concentration was determined using the Bradford method. Equal amounts (40 μg) of protein lysates extracted from macrophages were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, MA, US). The membranes were blocked with 5% fat-free skim milk in Tris-buffered saline containing 0.5% Tween-20 (TBS-T, pH 7.4) at room temperature for 1 h. After blotting, the membranes were incubated for 12 h at 4 °C with the appropriate primary antibodies. The membranes were washed three times with TBS-T buffer at 5 min intervals. The blots were then incubated with secondary HRPconjugated goat anti-rabbit IgG antibodies and washed three times in TBS-T. The blots were visualised using an ECL Western blotting detection system (Beyotime Institute of Biotechnology, Haimen, China) and exposed to an X-ray film. β-actin was used as a loading control for the whole-cell extracts. Each band was quantitatively evaluated using the ImageJ software package.
2.7. Total RNA extraction and real-time quantitative PCR Total RNA was isolated from the macrophages using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The reverse transcription step was carried out using the RevertAid™ First Strand cDNA Synthesis Kit (Fermentas, Ontario, Canada) to synthesise cDNA. The quantitative PCR analysis was performed with a SLAN real time PCR detection system. The PCR thermocycling parameters were 95 °C for 1 min followed by 40 cycles of 95 °C for 15 s, 58 °C for 25 s, and 72 °C for 20 s. The amplification was completed with a final extension step at 72 °C for 5 min. The relative gene expression (RFR) was calculated by the comparative Ct method (RFR = 2−△△CT). Each sample was run in duplicate and all of the samples were analysed in parallel for the expression of the housekeeping gene β-actin, which was used as an endogenous control for normalisation of the expression level of target genes. The fold induction was determined from the mean replicate values. The primer sequences for the analysis of NOS-2, COX-2, TNF-α, IL-6 and β-actin mRNA are described in Supplementary Table 1.
2.8. LPS-induced endotoxic shock and measurement of TNF-α and IL-6 levels Twenty-four mice were randomly divided into two groups (12 mice per group). The groups of mice were injected i.p. with PBS (control), GF5 (10, 30 and 90 mg/kg) or Indomethacin (8 mg/kg). One hour after administration, the control, GF5 and Indomethacin-administered mice were injected i.p. with 15 mg/kg of LPS. The survival of both groups was monitored for 7 days. At 90 min after the injection of LPS, blood was collected by submandibular bleeding to evaluate the changes in the serum levels of TNF-α and IL-6. This time point was chosen because 90 min of endotoxemic exposure results in a maximal increase in the serum levels of TNF-α in this species [19]. The blood sample was centrifuged (3300 ×g for 3 min) to separate the serum. The content of TNF-α and IL-6 in the serum samples (50 μl) was determined by enzymelinked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's protocol.
2.9. Statistical analysis Fig. 4. The dose-dependent inhibitory effect of GF5 on NO and PGE2 release. (A) Peritoneal macrophages were pre-incubated for 30 min with different concentrations of GF5 (0.1, 1,10, 20, 50 and 100 μM) followed by stimulation with 100 ng/ml LPS for 24 h. (B) Peritoneal macrophages were treated as in A, and PGE2 release was measured with an ELISA kit. The results shown are the means ± S.D. of three independent experiments. *P b 0.05, **P b 0.01, ***P b 0.001, with respect to the LPS condition.
The data shown are means ± S.D. of at least three separate experiments. Significant differences were established by one-way ANOVA using SPSS 16.0 software. The Kaplan–Meier method was used to compare the differences in mortality rates between groups. P b 0.05 was accepted as statistically significant.
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Fig. 5. NOS-2 and COX-2 expression levels are reduced by GF5. (A) Macrophages were pre-incubated for 30 min with GF5 (0.1, 1, 10 and 50 μM) followed by stimulation with 100 ng/ml LPS for 24 h. NOS-2 and COX-2 proteins were detected by Western blotting. β-actin content was used as a loading control. Western blot shows a representative experiment of three. (B) Densitometry analysis of NOS-2 and COX-2 expression was performed. The results shown are the means ± S.D. of three independent Western blot experiments and are expressed as the relative signal intensity compared with the LPS condition. *P b 0.05, **P b 0.01, ***P b 0.001. (C) The macrophages were pre-incubated for 30 min with GF5 (0.1, 1, 10 and 50 μM) followed by stimulation with 100 ng/ml LPS for 4 h. The relative expression levels of NOS-2 and COX-2 were determined by real-time PCR. The results shown are the means ± S.D. of three independent experiments. **P b 0.01, ***P b 0.001 compared with the LPS condition.
3. Results
3.3. GF5 inhibits NO and PGE2 release in LPS-stimulated macrophages
3.1. Effects of GF5 on cell viability
To investigate the inhibitory effects of GF5 on the production of relevant inflammatory mediators, such as NO and PGE2, peritoneal macrophages were pre-treated with GF5 for 30 min and subsequently incubated with LPS for 24 h. As shown in Fig. 4, GF5 treatment suppressed NO production in a dose-dependent manner (IC50 = 3.84 μM). Similar results were obtained for PGE2 release (IC50 = 3.16 μM).
We analysed cell viability percentage using a colorimetric cell counting kit after incubating cells for 24 h in the absence or presence of LPS. The cell viabilities were not affected by GF5 in the concentration up to 100 μM (Fig. 2). Thus, the effects of GF5 on macrophages were not attributable to its cytotoxic effects.
3.4. GF5 inhibits the induction of NOS-2 and COX-2 at the transcriptional level 3.2. GF5 inhibits cytokine release To examine the effects of GF5 on LPS-induced inflammatory-related cytokine production, peritoneal macrophages were treated with various concentrations of GF5 for 30 min followed by LPS stimulation (100 ng/ml) for 4 h in mRNA level assay, as well as 24 h in protein level assay. The expression levels of TNF-α and IL-6 following LPS induction were evaluated by ELISA and real-time PCR. The results showed that GF5 treatment led to a dose-dependent suppression of TNF-α and IL-6 protein (IC50 = 4.09 μM and 17.04, Fig. 3.A) and mRNA (Fig. 3.B) levels in LPS-stimulated macrophages.
To further analyse the signalling pathways modulated by GF5, we asked whether its inhibitory effects on the pro-inflammatory mediators PGE2 and NO correlated with NOS-2 and COX-2 enzyme modulation. Fig. 5 shows that GF5 inhibited LPS-induced NOS-2 and COX-2 mRNA expression, as measured by real-time PCR. 3.5. Anti-inflammatory effects of GF5 are mediated by NF-κB inhibition Activation of NF-κB is a key step in the onset of the inflammatory response. Many intracellular messengers are involved in the NF-κB signalling pathway. Among them, IκB-α, one of the most important inhibitors
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Fig. 6. GF5 inhibits NF-κB activity. (A) The macrophages were pre-treated for 30 min with GF5 (10 μM) and then activated for the indicated times (15, 30, 60 and 90 min) with 100 ng/ml LPS. (B) The cells were pre-treated for 30 min with GF5 (1, 10 and 50 μM) and then activated for 30 min with 100 ng/ml LPS. The levels of p-IκB-α, IκB-α, p-IκB-β, IκBβ, p-p65 and p-65 were measured using Western blot analysis. β-actin was used as a loading control for extracts. A representative blot is shown along with the mean ± S.D. from densitometry analysis of three independent experiments. *P b 0.05, **P b 0.01.
of the NF-κB complex, inactivates NF-κB by binding to it in the cytosol [20]. To test whether the GF5-dependent inhibition of the inflammatory response is mediated through the NF-κB pathway, NF-κB, IκB-α and IκB-β levels were determined by Western blot. Phosphorylations of IκB-α and IκB-β were impaired in activated cells that had been pretreated with GF5 (Fig. 6A, B). Compared with IκB-β, GF5 exerts more effect on phosphorylations of IκB-α, especially at high concentration (50 μM). This effect was accompanied by an inhibition of IκB-α and IκB-β degradation and a reduced accumulation of the NF-κB p65 subunit in the nucleus after LPS stimulation.
3.6. Effects of GF5 on MAPK activation The phosphorylation and activation status of kinases in the MAPK system has a crucial impact on cytokine production. MAPKs, including ERK, JNK and p38, also play essential roles in the regulation of proinflammatory cytokine production [21]. To determine whether the MAPK pathway mediates the inhibition of the GF5-dependent inflammatory response, the activation states of JNK, ERK and P38 MAPKs were examined by Western blot. As is shown in Fig. 7, GF5 significantly suppressed the phosphorylation of JNK and p38 MAPK, whereas ERK
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Fig. 7. Regulation of MAPK activities by GF5. (A) The macrophages were pre-treated for 30 min with GF5 (10 μM) and then activated for the indicated times (15, 30, 60 and 90 min) with 100 ng/ml LPS. (B) The cells were pre-treated for 30 min with GF5 (1, 10 and 50 μM) and then activated for 30 min with 100 ng/ml LPS. The levels of p-JNK, JNK, p-p38 and p38 were measured using Western blot analysis. β-actin was used as a loading control. A representative blot is shown along with the mean ± S.D. from densitometry analysis of three independent experiments. *P b 0.05, **P b 0.01.
phosphorylation was not substantially affected. These results show that MAPK signal transduction may be effectively blocked by GF5 treatment in stimulated macrophages.
production of pro-inflammatory cytokines in vivo, similar to its inhibitory effect in vitro. 4. Discussion and conclusion
3.7. GF5 administration improves survival and attenuates the inflammatory response in endotoxemic animals To further confirm the activity of GF5 in vivo, mice were injected with LPS in the presence of GF5. The results showed that mice pretreated with GF5 (10, 30 and 90 mg/kg, i.p.) had higher survival rates compared with untreated mice (47.62, 76.15 and 83.08% vs. 0%, Fig. 8A). The overall differences in survival rates between the groups with and without GF5 (30 and 90 mg/kg) were significant (P b 0.05 and P b 0.01), suggesting that GF5 protects mice from sepsis-induced death. Furthermore, improved survival was associated with decreased serum levels of TNF-α and IL-6 in GF5-treated mice compared with untreated mice (Fig. 8B). These results demonstrate that GF5 decreases the
Terpenoids, a large and widely occurring class of organic compounds, have been found in higher plants, algae, mosses, liverworts and lichens, microbes, insects and marine organisms [22–27]. Labdanes, members of the bicyclic diterpenoid group, have been identified as having a wide spectrum of biological activities [28–31], but few reports have examined the potential anti-inflammatory actions of labdane diterpene glycosides and the mechanisms involved. Our research has demonstrated that GF5, an ent-labdane diterpene glycoside, effectively decreased the production of NO and PGE2 in a dose-dependent manner and that this inhibitory effect was not mediated by cytotoxic effects in LPS-treated macrophages. This inhibition was accompanied by a concentration-dependent down-regulation in the
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induced mice. These results suggest that GF5 possesses useful antiinflammatory activity by inhibiting the production of inflammatory mediators and cytokines. Our research also found that GF5 decreased the phosphorylations of IκB-α, IκB-β and NF-κB p65 protein, while suppressed the phosphorylations of JNK and p38 but not ERK in LPSstimulated cells in a dose-dependent manner. Thus, GF5 suppressed LPS-induced pro-inflammatory cytokine and inflammatory mediator production in macrophages by preventing NF-κB and MAPK pathway activation. GF5, the ent-labdane diterpene glucoside, is another class of compound compared to the classical nonsteroidal anti-inflammatory drugs such as indomethacin and dexamethasone. The low cytotoxicity of this compound in cell culture and their effectiveness in an animal model of inflammation suggest that GF5 has potential use in the treatment of inflammatory diseases. In conclusion, this study demonstrated that GF5 has antiinflammatory activities in vitro and in vivo. The effects are mediated by the inhibition of the NF-κB and MAPK signalling pathways. These data may provide new insights into the mechanism for the antiinflammatory activity of ent-labdane diterpene glucoside and suggest that ent-labdane diterpene glucoside may be the antiinflammatory component of Rubi Fructus and constitute a valid pharmacological approach in the treatment of inflammatory disease. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.intimp.2014.12.007.
References
Fig. 8. GF5 increased the survival rate and reduced the circulating levels of IL-6 and TNF-α in a LPS-endotoxemia model. Mice were injected i.p. with 15 mg/kg LPS (n = 12), 15 mg/kg LPS and GF5 (10, 30 and 90 mg/kg, n = 12) or 15 mg/kg LPS and indomethacin (8 mg/kg, n = 12). Their survival was monitored at intervals of 12 h for 7 days. (A) The overall difference in survival rate between the groups with and without GF5. (B) Serum levels of TNF-α and IL-6 were assayed using ELISA kits. The results are expressed as the mean ± S.D. *P b 0.05 and **P b 0.01.
expression of NOS-2 and COX-2 at both the protein and mRNA levels in LPS-treated macrophages. GF5 inhibited the production of TNF-α and IL-6 in vitro and in vivo and improved survival following sepsis in LPS-
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Suppression of nuclear factor-kappa B and mitogen-activated protein kinase signalling pathways by goshonoside-F5 extracted from Rubi Fructus Jian-Ming He a,b,1, Shi-Cai Chen c,1, Run-Ping Li d, Liang-Xi Yuan e, Jun-Min Bao e,⁎, Mei-Li Guo a,⁎⁎ a
Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai 200433, China School of Pharmacy, Fudan University, Shanghai 201203, China c Department of Otorhinolaryngology, Changhai Hospital, Shanghai 200433, China d Department of Diving Medicine, Faculty of Navy Medicine, Second Military Medical University, Shanghai 200433, China e Department of Vascular Surgery, Changhai Hospital, Second Military Medical University, Shanghai 200433, China b
a r t i c l e
i n f o
Article history: Received 19 September 2014 Received in revised form 3 November 2014 Accepted 3 December 2014 Available online 15 December 2014 Keywords: Rubi Fructus Ent-labdane diterpene glucoside Macrophage Anti-inflammatory NF-κB MAPK
a b s t r a c t Rubi Fructus, a traditional Chinese medicine, was considered as an anti-inflammatory agent in folk medicine. In the present study, we investigated the signalling pathways involved in the anti-inflammatory effects of goshonoside-F5 (GF5), isolated from Rubi Fructus, in peritoneal macrophages and examined its therapeutic effect in a mouse endotoxic shock model. GF5 decreased NO and PGE2 production in LPS-stimulated macrophages (IC50 = 3.84 and 3.16 μM). This effect involved the suppression of NOS-2 and COX-2 gene expression at the transcriptional level. Examination of the effects of GF5 on NF-κB signalling demonstrated that it inhibits the phosphorylation of IκB-α and IκB-β, blocking their degradation and the nuclear translocation of the NF-κB p65 subunit. Moreover, inhibition of MAPK signalling was also observed, and phosphorylation of p38 and JNK was suppressed in the presence of GF5. Inflammatory cytokines, including IL-6 and TNF-α, were down-regulated by this compound after activation with LPS (IC50 = 17.04 and 4.09 μM). Additionally, GF5 (30 and 90 mg/kg, i.p.) significantly reduced the circulating cytokine levels (IL-6 and TNF-α) and increased survival in a mouse model of endotoxemia. These results show that GF5 significantly inhibits the pro-inflammatory response induced by LPS, both in vitro and in vivo. Our results provide a strong pharmacological basis for further understanding the potential therapeutic role of GF5 in inflammatory disease and shed new light on the bioactivity of ent-labdane diterpene glucoside. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Inflammation is a host response by an organism to foreign pathogens or tissue injury to eliminate harmful stimuli, including infection and noxious stimuli, as well as to promote healing and repair processes in the damaged tissue [1]. Macrophages play a key role in the acute immune response by recognising and phagocytosing pathogens, releasing cytokines and chemokines, processing antigens and repairing tissue damage [2]. Upon the activation of Toll Like Receptor 4 (TLR4) by LPS in macrophages, MAPKs become phosphorylated and also phosphorylate the IκB kinase (IKK) complex, eventually activating the NF-κB and MAPK pathways, resulting in the production of NO, PGE2 and various cytokines, such as TNF-α, IL-1β and IL-6. NO and PGE2 are generated in
⁎ Corresponding author. Tel.: +86 21 81861566. ⁎⁎ Corresponding author. Tel./fax: +86 21 81871302. E-mail addresses: [email protected] (S.-C. Chen), [email protected] (J.-M. Bao), [email protected] (M.-L. Guo). 1 Co-first author.
http://dx.doi.org/10.1016/j.intimp.2014.12.007 1567-5769/© 2014 Elsevier B.V. All rights reserved.
macrophages by the inducible isoforms of cyclooxygenase (COX-2) and NO synthase (NOS-2), respectively. These pro-inflammatory cytokines and signalling molecules play important roles in inflammatory diseases and pathological conditions [3–7]. Rubus chingii Hu, a member of the Rosaceae family, is extensively grown in both north and south China. Rubi Fructus, the dried unripe fruit of R. chingii, has been used as a food and tonic for thousands of years. In traditional Chinese medicine, this fruit is proposed to give tone to the kidney and liver [8]. Moreover, it is considered as an antiinflammatory agent in folk medicine to treat diseases such as urinary tract infections, nephritis, chronic hepatitis and enteritis. In modern pharmacological experiments, the anti-oxidant [9], hepatoprotective [10], antifatigue [11] and immunomodulatory [12] properties of Rubi Fructus have been reported, and it has additional effects on the hypothalamus–pituitary–gonad axis [13]. However, the anti-inflammatory compounds derived from Rubi Fructus have rarely been reported. Our previous phytochemistry study of Rubi Fructus resulted in the isolation of various compounds classified as flavonoids (isoquercetin, kaempferol-3-O-β-Drutinoside, tiliroside and kaempferol), benzaldehydes and organic
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Fig. 1. Chemical structure and HPLC chromatogram of GF5.
acids (vanillic aldehyde, 4-Hydroxy-3-methoxybenzoic acid and pHydroxybenzaldehyde), steroids (β-sitosterol, Stigmast-5-en-3-ol, Oleate), and ent-labdane diterpenes (goshonoside-G, goshonoside-F2, goshonoside-F4 and goshonoside-F5) [14,15]. We reported the potential anti-inflammatory activity of the novel compound goshonoside-G [15]. However, goshonoside-F5 (GF5) is also a major compound (Fig. 1) in Rubi Fructus. The present study was designed to investigate the antiinflammatory effects of GF5 in LPS-activated macrophages and in mice undergoing endotoxic shock. We further examined whether GF5 could reduce inflammatory responses through the suppression of the NF-κB Table 1 NMR data (δ in ppm) of GF5 (C5D5N). Position
GF5 δH (J in Hz)
δC
1.20 (m), 1.77 (m) 1.64 (m) 3.75 (1H, t, J = 8 Hz)
5.32 (1H, t, J = 7 Hz) 4.31 (1H, s), 4.82 (1H, s) 1.68 (3H, s) 4.52 (1H, s), 4.82 (1H, s) 3.25, 3.81 (1H, d, J = 9.5) 0.73 (3H, s) 0.68 (3H, s)
38.4 28.2 73.3 44.2 48.0 25.3 39.2 150.1 57.1 40.5 23.2 39.7 142.9 121.8 66.6 16.8 107.2 74.4 13.0 15.8
15-O-Glc 1′ 2′ 3′ 4′ 5′ 6′
4.29 (1H, d, J = 8 Hz) 3.22 (m) 3.26 (m) 3.30 (m) 3.35 (m) 3.64 (m), 3.84 (m)
103.0 75.2 78.1 72.0 78.5 63.1
18-O-Glc 1″ 2″ 3″ 4″ 5″ 6″
4.22 (1H, d, J = 8 Hz) 3.18 (m) 3.27 (m) 3.26 (m) 3.35 (m) 3.64 (m), 3.84 (m)
105.0 75.3 78.3 72.2 78.6 63.2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1.85 (m) 1.35 (m), 1.70 (m) 2.07 (m), 2.33 (m) 1.82 (m) 1.46 (m), 1.62 (m) 1.88 (m), 2.15 (m)
and MAPK signalling pathways. The following is our first report of this study. 2. Materials and methods 2.1. Materials ESI-MS was measured on a Varian MAT-212 mass spectrometer. Silica gel (200–300 mesh, Qingdao Haiyang Chemical Co. Ltd., Qingdao, China). The 1H NMR and 13C NMR spectra were recorded on a Bruker DMX-400 NMR spectrometer with tetramethylsilane (TMS) as an internal standard. HPLC assays were conducted using an Agilent 1100 HPLC (Agilent Technologies, Palo Alto, CA, USA). Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FBS) and penicillin–streptomycin solution were obtained from Gibco (Carlsbad, CA, USA). Lipopolysaccharide (LPS) (Escherichia coli strain 0111:B4) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for phospho-IκB-α, phospho-IκB-β, IκB-α, IκB-β, p-65, phospho-p38, p38, phospho-JNK and COX-2 were obtained from EPITOMICS (San Diego, CA, USA). Antibodies against phospho-p42–p44 ERK, p42–p44 ERK and COX-2 were purchased from Cell Signalling Technology (Danvers, MA, USA). Anti-JNK, anti-β-actin and anti-NOS-2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Goat anti-rabbit IgG-HRP secondary antibodies and Cy3 goat anti-rabbit antibodies were from KPL (Gaithersburg, MD, USA). Enzyme linked immunosorbent assay (ELISA) kits for PGE2, TNF-α and IL-6 were from R&D Systems (Minneapolis, MN, USA). Male C57BL/6Slac mice (6–8 weeks old) were obtained from SLRC Laboratory Animal Co., Ltd. (Shanghai, China). All procedures were conducted in accordance with the University guidelines and approved by the Ethical Committee for Animal Care and the Use of Laboratory Animals at Second Military Medical University. C57BL/6Slac mice free of pathogens were bred in our animal facility and supplied with food and water ad libitum and exposed to a 12 h light–dark cycle. 2.2. Preparation of GF5 and its purity 2.2.1. Isolation of GF5 from Rubi Fructus Dry Rubi Fructus (1000 g) was extracted three times using 70% ethanol as a solvent. The solvent was evaporated under reduced pressure to give an ethanol extract, which was further subjected to chromatography using a column packed with macroporous resin D101 (Chemical Plant of NanKai University, Tianjin, China) and eluted with water and 30%, 60%, and 95% aqueous ethanol. The 60% ethanol extract (130 g) was further fractionated by MCI-gel CHP20P (Mitsubishi Chemical Co., Tokyo) column chromatography using a gradient of aqueous ethanol
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2.2.2. High-performance liquid chromatography (HPLC) The purity of GF5 was determined by high performance liquid chromatography (HPLC) using an Agilent Series 1100 liquid chromatograph coupled with evaporative light scattering detection (ELSD). Chromatography was performed through an Agilent Zorbax-C18 column (5 μm, 250 mm × 4.6 mm, i.d.), which was controlled at 35 °C using acetonitrile (A) and H2O (B) as the mobile phase under a gradient programme with a flow rate of 1.0 ml/min: 0–5 min, 75–69% A; 5–12 min, 69–65% A. The ELSD was programmed with the following settings: evaporator temperature, 40 °C; air pressure, 3.5 bar. The purity of goshonoside-F5 was determined to be 98.7% by normalisation of the peak areas detected by HPLC (Fig. 1). 2.3. Preparation of peritoneal macrophages
Fig. 2. Effects of GF5 on cell viability. Peritoneal macrophages were incubated with different concentrations of GF5 (0.1, 1,10, 20, 50 and 100 μM) in the presence or absence of 100 ng/ml LPS for 24 h. Cell viability was determined by the CCK-8 assay. The results shown are the means ± S.D. of three independent experiments.
(20, 30, 40, 50, 60, 80 and 100%) to give a 60% ethanol fraction. The 60% ethanol fraction was then separated using Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden) column chromatography and eluted with EtOH–H2O (2:5 v/v) to yield the investigated compound in present study (white powder, 487 mg), which was stained purple with ethanolic vanillin–sulphuric acid. The structure of this compound was further identified using characteristic and spectral methods. The ESI mass spectrum in positive mode showed quasimolecular ion peaks at m/z 669 [M + Na]+, indicating a molecular mass (M 664) corresponding to the formula C32H54O13. The structure of this compound was determined as goshonoside-F5 (GF5), and spectroscopic data was shown in Table 1, which agrees with spectra in the literature [16].
Peritoneal macrophages were isolated from the C57BL/6Slac mice essentially as previously described [17], with slight modification. Briefly, the mice were intraperitoneally (i.p.) injected with sterilised culture broth (1 mL). On day 3 following the injection, the peritoneal exudate cells were isolated by washing the peritoneal cavity with ice-cold PBS. The cells were seeded at 1 × 106/cm2 in DMEM supplemented with 10% FBS and 2% penicillin–streptomycin antibiotic at 37 °C in a 5% CO2 incubator. Two hours later, the culture medium was replaced to remove the non-adherent cells. The adherent cells were incubated at 37 °C in a humidified 5% CO2 atmosphere overnight and used as peritoneal macrophages for the subsequent procedures. 2.4. Assay for cell viability Macrophages were plated at a density of 105 cells/well in 96-well plates. To determine the appropriate non-toxic treatment concentrations, cells were incubated in the presence of different concentrations of GF5 for 24 h. The viable cell content was measured using colorimetric cell counting kit 8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan). The
Fig. 3. Cytokine release is inhibited by GF5. (A) Macrophages were treated with GF5 (0.1, 1, 10, and 50 μM) or a control treatment for 30 min followed by 4 h of stimulation with 100 ng/ml LPS. mRNA levels of IL-6 and TNF-α were determined by real-time PCR. (B) Macrophages were treated with GF5 (0.1, 1, 10, 20, 50 and 100 μM) or a control treatment for 30 min followed by 24 h of stimulation with 100 ng/ml LPS. The production of IL-6 and TNF-α was determined in the supernatant by ELISA. The results shown are the means ± S.D. of three independent experiments carried out in triplicate. *P b 0.05, **P b 0.01, ***P b 0.001 vs. LPS.
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assays were performed in triplicate, and the results are expressed as the percent reduction in cell viability compared with untreated control cultures for at least three independent experiments.
2.5. Assay of inflammatory mediator and cytokine release 2.5.1. Determination of NO biosynthesis The nitric oxide (NO) content of the culture supernatant was measured using the Griess Reagent (Beyotime Institute of Biotechnology, Jiangsu, China) by determining the concentration of NaNO2 produced by NO metabolism [18].
2.5.2. Assay of PGE2 release The levels of PGE2 in the culture supernatant were quantified using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.
2.5.3. Cytokine assay Cytokine (IL-6 and TNF-α) production by cultured macrophages was determined using an enzyme-linked immunosorbent assay (ELISA) kit from R&D systems according to the manufacturer's instructions.
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2.6. Western blot analysis The treated cells were washed twice with ice-cold PBS. Total cellular protein was extracted using the Protein Extraction Reagent (Goodbio, Wuhan, China). Protein concentration was determined using the Bradford method. Equal amounts (40 μg) of protein lysates extracted from macrophages were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, MA, US). The membranes were blocked with 5% fat-free skim milk in Tris-buffered saline containing 0.5% Tween-20 (TBS-T, pH 7.4) at room temperature for 1 h. After blotting, the membranes were incubated for 12 h at 4 °C with the appropriate primary antibodies. The membranes were washed three times with TBS-T buffer at 5 min intervals. The blots were then incubated with secondary HRPconjugated goat anti-rabbit IgG antibodies and washed three times in TBS-T. The blots were visualised using an ECL Western blotting detection system (Beyotime Institute of Biotechnology, Haimen, China) and exposed to an X-ray film. β-actin was used as a loading control for the whole-cell extracts. Each band was quantitatively evaluated using the ImageJ software package.
2.7. Total RNA extraction and real-time quantitative PCR Total RNA was isolated from the macrophages using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The reverse transcription step was carried out using the RevertAid™ First Strand cDNA Synthesis Kit (Fermentas, Ontario, Canada) to synthesise cDNA. The quantitative PCR analysis was performed with a SLAN real time PCR detection system. The PCR thermocycling parameters were 95 °C for 1 min followed by 40 cycles of 95 °C for 15 s, 58 °C for 25 s, and 72 °C for 20 s. The amplification was completed with a final extension step at 72 °C for 5 min. The relative gene expression (RFR) was calculated by the comparative Ct method (RFR = 2−△△CT). Each sample was run in duplicate and all of the samples were analysed in parallel for the expression of the housekeeping gene β-actin, which was used as an endogenous control for normalisation of the expression level of target genes. The fold induction was determined from the mean replicate values. The primer sequences for the analysis of NOS-2, COX-2, TNF-α, IL-6 and β-actin mRNA are described in Supplementary Table 1.
2.8. LPS-induced endotoxic shock and measurement of TNF-α and IL-6 levels Twenty-four mice were randomly divided into two groups (12 mice per group). The groups of mice were injected i.p. with PBS (control), GF5 (10, 30 and 90 mg/kg) or Indomethacin (8 mg/kg). One hour after administration, the control, GF5 and Indomethacin-administered mice were injected i.p. with 15 mg/kg of LPS. The survival of both groups was monitored for 7 days. At 90 min after the injection of LPS, blood was collected by submandibular bleeding to evaluate the changes in the serum levels of TNF-α and IL-6. This time point was chosen because 90 min of endotoxemic exposure results in a maximal increase in the serum levels of TNF-α in this species [19]. The blood sample was centrifuged (3300 ×g for 3 min) to separate the serum. The content of TNF-α and IL-6 in the serum samples (50 μl) was determined by enzymelinked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's protocol.
2.9. Statistical analysis Fig. 4. The dose-dependent inhibitory effect of GF5 on NO and PGE2 release. (A) Peritoneal macrophages were pre-incubated for 30 min with different concentrations of GF5 (0.1, 1,10, 20, 50 and 100 μM) followed by stimulation with 100 ng/ml LPS for 24 h. (B) Peritoneal macrophages were treated as in A, and PGE2 release was measured with an ELISA kit. The results shown are the means ± S.D. of three independent experiments. *P b 0.05, **P b 0.01, ***P b 0.001, with respect to the LPS condition.
The data shown are means ± S.D. of at least three separate experiments. Significant differences were established by one-way ANOVA using SPSS 16.0 software. The Kaplan–Meier method was used to compare the differences in mortality rates between groups. P b 0.05 was accepted as statistically significant.
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Fig. 5. NOS-2 and COX-2 expression levels are reduced by GF5. (A) Macrophages were pre-incubated for 30 min with GF5 (0.1, 1, 10 and 50 μM) followed by stimulation with 100 ng/ml LPS for 24 h. NOS-2 and COX-2 proteins were detected by Western blotting. β-actin content was used as a loading control. Western blot shows a representative experiment of three. (B) Densitometry analysis of NOS-2 and COX-2 expression was performed. The results shown are the means ± S.D. of three independent Western blot experiments and are expressed as the relative signal intensity compared with the LPS condition. *P b 0.05, **P b 0.01, ***P b 0.001. (C) The macrophages were pre-incubated for 30 min with GF5 (0.1, 1, 10 and 50 μM) followed by stimulation with 100 ng/ml LPS for 4 h. The relative expression levels of NOS-2 and COX-2 were determined by real-time PCR. The results shown are the means ± S.D. of three independent experiments. **P b 0.01, ***P b 0.001 compared with the LPS condition.
3. Results
3.3. GF5 inhibits NO and PGE2 release in LPS-stimulated macrophages
3.1. Effects of GF5 on cell viability
To investigate the inhibitory effects of GF5 on the production of relevant inflammatory mediators, such as NO and PGE2, peritoneal macrophages were pre-treated with GF5 for 30 min and subsequently incubated with LPS for 24 h. As shown in Fig. 4, GF5 treatment suppressed NO production in a dose-dependent manner (IC50 = 3.84 μM). Similar results were obtained for PGE2 release (IC50 = 3.16 μM).
We analysed cell viability percentage using a colorimetric cell counting kit after incubating cells for 24 h in the absence or presence of LPS. The cell viabilities were not affected by GF5 in the concentration up to 100 μM (Fig. 2). Thus, the effects of GF5 on macrophages were not attributable to its cytotoxic effects.
3.4. GF5 inhibits the induction of NOS-2 and COX-2 at the transcriptional level 3.2. GF5 inhibits cytokine release To examine the effects of GF5 on LPS-induced inflammatory-related cytokine production, peritoneal macrophages were treated with various concentrations of GF5 for 30 min followed by LPS stimulation (100 ng/ml) for 4 h in mRNA level assay, as well as 24 h in protein level assay. The expression levels of TNF-α and IL-6 following LPS induction were evaluated by ELISA and real-time PCR. The results showed that GF5 treatment led to a dose-dependent suppression of TNF-α and IL-6 protein (IC50 = 4.09 μM and 17.04, Fig. 3.A) and mRNA (Fig. 3.B) levels in LPS-stimulated macrophages.
To further analyse the signalling pathways modulated by GF5, we asked whether its inhibitory effects on the pro-inflammatory mediators PGE2 and NO correlated with NOS-2 and COX-2 enzyme modulation. Fig. 5 shows that GF5 inhibited LPS-induced NOS-2 and COX-2 mRNA expression, as measured by real-time PCR. 3.5. Anti-inflammatory effects of GF5 are mediated by NF-κB inhibition Activation of NF-κB is a key step in the onset of the inflammatory response. Many intracellular messengers are involved in the NF-κB signalling pathway. Among them, IκB-α, one of the most important inhibitors
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Fig. 6. GF5 inhibits NF-κB activity. (A) The macrophages were pre-treated for 30 min with GF5 (10 μM) and then activated for the indicated times (15, 30, 60 and 90 min) with 100 ng/ml LPS. (B) The cells were pre-treated for 30 min with GF5 (1, 10 and 50 μM) and then activated for 30 min with 100 ng/ml LPS. The levels of p-IκB-α, IκB-α, p-IκB-β, IκBβ, p-p65 and p-65 were measured using Western blot analysis. β-actin was used as a loading control for extracts. A representative blot is shown along with the mean ± S.D. from densitometry analysis of three independent experiments. *P b 0.05, **P b 0.01.
of the NF-κB complex, inactivates NF-κB by binding to it in the cytosol [20]. To test whether the GF5-dependent inhibition of the inflammatory response is mediated through the NF-κB pathway, NF-κB, IκB-α and IκB-β levels were determined by Western blot. Phosphorylations of IκB-α and IκB-β were impaired in activated cells that had been pretreated with GF5 (Fig. 6A, B). Compared with IκB-β, GF5 exerts more effect on phosphorylations of IκB-α, especially at high concentration (50 μM). This effect was accompanied by an inhibition of IκB-α and IκB-β degradation and a reduced accumulation of the NF-κB p65 subunit in the nucleus after LPS stimulation.
3.6. Effects of GF5 on MAPK activation The phosphorylation and activation status of kinases in the MAPK system has a crucial impact on cytokine production. MAPKs, including ERK, JNK and p38, also play essential roles in the regulation of proinflammatory cytokine production [21]. To determine whether the MAPK pathway mediates the inhibition of the GF5-dependent inflammatory response, the activation states of JNK, ERK and P38 MAPKs were examined by Western blot. As is shown in Fig. 7, GF5 significantly suppressed the phosphorylation of JNK and p38 MAPK, whereas ERK
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Fig. 7. Regulation of MAPK activities by GF5. (A) The macrophages were pre-treated for 30 min with GF5 (10 μM) and then activated for the indicated times (15, 30, 60 and 90 min) with 100 ng/ml LPS. (B) The cells were pre-treated for 30 min with GF5 (1, 10 and 50 μM) and then activated for 30 min with 100 ng/ml LPS. The levels of p-JNK, JNK, p-p38 and p38 were measured using Western blot analysis. β-actin was used as a loading control. A representative blot is shown along with the mean ± S.D. from densitometry analysis of three independent experiments. *P b 0.05, **P b 0.01.
phosphorylation was not substantially affected. These results show that MAPK signal transduction may be effectively blocked by GF5 treatment in stimulated macrophages.
production of pro-inflammatory cytokines in vivo, similar to its inhibitory effect in vitro. 4. Discussion and conclusion
3.7. GF5 administration improves survival and attenuates the inflammatory response in endotoxemic animals To further confirm the activity of GF5 in vivo, mice were injected with LPS in the presence of GF5. The results showed that mice pretreated with GF5 (10, 30 and 90 mg/kg, i.p.) had higher survival rates compared with untreated mice (47.62, 76.15 and 83.08% vs. 0%, Fig. 8A). The overall differences in survival rates between the groups with and without GF5 (30 and 90 mg/kg) were significant (P b 0.05 and P b 0.01), suggesting that GF5 protects mice from sepsis-induced death. Furthermore, improved survival was associated with decreased serum levels of TNF-α and IL-6 in GF5-treated mice compared with untreated mice (Fig. 8B). These results demonstrate that GF5 decreases the
Terpenoids, a large and widely occurring class of organic compounds, have been found in higher plants, algae, mosses, liverworts and lichens, microbes, insects and marine organisms [22–27]. Labdanes, members of the bicyclic diterpenoid group, have been identified as having a wide spectrum of biological activities [28–31], but few reports have examined the potential anti-inflammatory actions of labdane diterpene glycosides and the mechanisms involved. Our research has demonstrated that GF5, an ent-labdane diterpene glycoside, effectively decreased the production of NO and PGE2 in a dose-dependent manner and that this inhibitory effect was not mediated by cytotoxic effects in LPS-treated macrophages. This inhibition was accompanied by a concentration-dependent down-regulation in the
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induced mice. These results suggest that GF5 possesses useful antiinflammatory activity by inhibiting the production of inflammatory mediators and cytokines. Our research also found that GF5 decreased the phosphorylations of IκB-α, IκB-β and NF-κB p65 protein, while suppressed the phosphorylations of JNK and p38 but not ERK in LPSstimulated cells in a dose-dependent manner. Thus, GF5 suppressed LPS-induced pro-inflammatory cytokine and inflammatory mediator production in macrophages by preventing NF-κB and MAPK pathway activation. GF5, the ent-labdane diterpene glucoside, is another class of compound compared to the classical nonsteroidal anti-inflammatory drugs such as indomethacin and dexamethasone. The low cytotoxicity of this compound in cell culture and their effectiveness in an animal model of inflammation suggest that GF5 has potential use in the treatment of inflammatory diseases. In conclusion, this study demonstrated that GF5 has antiinflammatory activities in vitro and in vivo. The effects are mediated by the inhibition of the NF-κB and MAPK signalling pathways. These data may provide new insights into the mechanism for the antiinflammatory activity of ent-labdane diterpene glucoside and suggest that ent-labdane diterpene glucoside may be the antiinflammatory component of Rubi Fructus and constitute a valid pharmacological approach in the treatment of inflammatory disease. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.intimp.2014.12.007.
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Fig. 8. GF5 increased the survival rate and reduced the circulating levels of IL-6 and TNF-α in a LPS-endotoxemia model. Mice were injected i.p. with 15 mg/kg LPS (n = 12), 15 mg/kg LPS and GF5 (10, 30 and 90 mg/kg, n = 12) or 15 mg/kg LPS and indomethacin (8 mg/kg, n = 12). Their survival was monitored at intervals of 12 h for 7 days. (A) The overall difference in survival rate between the groups with and without GF5. (B) Serum levels of TNF-α and IL-6 were assayed using ELISA kits. The results are expressed as the mean ± S.D. *P b 0.05 and **P b 0.01.
expression of NOS-2 and COX-2 at both the protein and mRNA levels in LPS-treated macrophages. GF5 inhibited the production of TNF-α and IL-6 in vitro and in vivo and improved survival following sepsis in LPS-
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