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NF-κB signaling pathway
International Journal of Biological Macromolecules 67 (2014) 330–335
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Lycium ruthenicum polysaccharide attenuates inflammation through inhibiting TLR4/NF- B signaling pathway Qiang Peng a,∗ , Huajing Liu b , Shihui Shi a , Ming Li c,∗ a b c
College of Food Science and Engineering, Northwest A&F University, Yangling 712100, China College of Resources and Environmental Science, Northeast Agricultural University, Harbin 150030, China College of Resources and Environment, Northwest A&F University, YangLing 712100, China
a r t i c l e
i n f o
Article history: Received 12 January 2014 Received in revised form 7 March 2014 Accepted 8 March 2014 Available online 25 March 2014 Keywords: Lycium ruthenicum Polysaccharide Toll-like receptor 4 NF- B
a b s t r a c t Polysaccharide has been reported to possess diverse biological activities, however, the inflammatory activity of polysaccharide isolated from Lycium ruthenicum remains unknown so far. In the present study, we investigated the effects of L. ruthenicum polysaccharide (LRGP3) on inflammatory reaction induced by lipopolysaccharide (LPS) in mouse macrophage RAW264.7 cells and some potential underlying mechanisms. Our results showed that LRGP3 treatment significantly inhibited the LPS-induced NO production and the mRNA expression of iNOS, as well as the level of Toll-like receptor 4 (TLR4). Furthermore, LRGP3 treatment prevented the I B␣ degradation and reduced phospho-NF- B p65 protein expression in LPSstimulated RAW264.7 cells. Meanwhile, the levels of pro-inflammatory cytokines, such as interleukin (IL)-␣, IL-6, tumor necrosis factor (TNF)-␣ were suppressed by LRGP3 in LPS-stimulated RAW264.7 cells. Taken together, our results suggested that LRGP3 attenuated LPS-induced inflammation via inhibiting TLR4/NF- B signaling pathway. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Inflammation, a physiological response to infection or injury, plays an important in various diseases, such as atherosclerosis, neurodegenerative disease, cancers. During the process of inflammation, different cell types are recruited, including monocytes that differentiate locally into macrophages. This leads to the regulated production of various pro- and antiinflammatory mediators including cytokines, such as interleukins (ILs), interferon, tumor necrosis factor (TNF)-␣ [1,2]. Previous studies showed that traditional Chinese medicine herbs possess anti-inflammatory property. Forsythiaside, the major bioactive component of Forsythia suspense Vahl., has been shown to exhibit a promising anti-inflammatory activity by decreasing the expression of pro-inflammatory cytokines in lipopolysaccharide (LPS)-induced inflammation on the bursa of Fabricius of chickens [3]. Ethanol extract from a traditional Chinese medicinal decoction,
∗ Corresponding authors at: Northwest A&F University, College of Food Science and Engineering, Xinong Road 22, Yangling, China. Tel.: +86 29 87091746/+86 451 87091793; fax: +86 29 87091746/+86 451 55190720. E-mail addresses: [email protected], [email protected] (Q. Peng), [email protected] (M. Li). http://dx.doi.org/10.1016/j.ijbiomac.2014.03.023 0141-8130/© 2014 Elsevier B.V. All rights reserved.
“Zuojin Pill”, inhibited the expression of inflammatory mediators in LPS-stimulated RAW264.7 mouse macrophages [4]. The phenolic glucoside, a main constituent of a Chinese herbal medicine, attenuated levels of neurotoxic proinflammatory mediators and proinflammatory cytokines in LPS-stimulated microglial cells [5]. Ginsenoside Rg1 also attenuated overactivation of microglial cells by repressing expression levels of neurotoxic proinflammatory cytokines in LPS-stimulated murine microglial cells [6]. Lycium ruthenicum Murr., belongs to the genus Lycium of the family Solanaceae, and is a native wild resource plant of China, found principally in Qinghai and Xinjiang Provinces. Active constituents of L. ruthenicum Murr. are reported to be have a variety of biological activities, including immunoregulation, anti-aging, lowering blood-sugar and blood-fat levels and anti-fatigue. Of these ingredients, polysaccharide has been known as the major bioactive compound. However, purified polysaccharide with antiinflammatory activity has not yet been documented. Recently, we have purified a water-soluble polysaccharide (LRGP3) from the crude polysaccharide extracted from L. ruthenicum Murr, and the structure of LRGP3 was shown in Fig. 1. LRGP3 was a highly branched polysaccharide with a backbone of (1 → 3)-linked -d-galactopyranosyl residues, many of which were substituted at the O-6 position. The branches were composed of (1 → 5)-linked arabinosyl, (1 → 2)-linked arabinosyl,
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Fig. 1. The structure of the repeat unit of the glycan of LRGP3.
(1 → 6)-linked galactosyl, (1 → 3)-linked galactosyl, and (1 → 2,4)linked rhamnosyl residues, and the major nonreducing termini were ␣-l-arabinofuranosyl residues [7]. In the present study, we investigated the anti-inflammatory activity of LRGP3 in LPS-stimulated mouse macrophage RAW264.7 cells by examining changes in levels of NO production and pro-inflammatory cytokines. Furthermore, the expression of Toll-like receptor 4 (TLR4) and downstream molecules of TLR4 signaling pathway were examined to investigate the potential mechanisms involved in the effects of LRGP3.
was recorded using a PR 4100 microplate reader (Bio-Rad, Bio-Rad, Hercules, CA, USA).
2. Materials and methods
Quantitative determination in the cell culture medium was made using the Enzyme-Linked Immunosorbent Assay (ELISA) kit (Invitrogen, Carlsbad, CA, USA) for IL-1␣, IL-6 and TNF-␣, according to manufacturer’s instruction, and the results are expressed in pg/mg of protein in each sample. All of the analyses were performed in triplicate.
2.1. Materials The anti-TLR4 antibody, anti-phospho-p65, anti-I B␣ and anti -actin antibodies were purchased from Invitrogen (Carlsbad, CA). All other chemicals and reagents were purchased from Sigma (Saint Louis, MO). 2.2. Preparation of LRGP3 The L. ruthenicum Murr. was extracted twice with boiling water for 2 h at a ratio of 20:1 (w/w) followed by centrifuging at 6000 rpm × 10 min. Supernatants were combined and concentrated to 1/5 of the original volume, and precipitated by adding 95% ethanol (4 vol). After centrifugation, the precipitate was dissolved with 500 mL of water and subjected to Savage method to remove free protein [8]. The de-proteinization solution was concentrated, dialyzed against distilled water, and lyophilized to obtain crude polysaccharides. The purity of CLRP was performed as previously described [7]. 2.3. Cell culture RAW264.7 mouse macrophage cells were obtained from American Type Cultured Collection (Rockville, MD, USA) and cultured in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum, glutamine, and antibiotics at 37 ◦ C under 5% CO2 . 2.4. Cell viability assay The viability of cells was measured using MTT assay. In brief, 5 × 103 cells/well RAW264.7 cells were plated in 96-well microplates overnight and then treated with different concentrations of LRGP3 or LPS (1 g/mL) for 24 h. Equal volume of medium was used as vehicle control. After treatment, cells were incubated with 0.5 mg/mL of MTT for another 4 h in dark and then the medium was discarded. The formazan crystals presented in cells were dissolved by 100 L dimethyl sulfoxide. The absorbance at 570 nm
2.5. NO production After preincubation of RAW264.7 cells for 18 h, cells were treated with LRGP3 (10–80 g/mL) or LPS (1 g/mL) for 24 h. Griess reagent was used to determine NO production [9]. 2.6. Quantitative analysis of cytokines
2.7. Real time-PCR Total RNA was extracted from RAW264.7 cells using TRIzol Reagent (Invitrogen). And mRNA was reversed transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Equal amounts of cDNA were submitted for PCR in the presence of SYBR Green I reagent and forward and reverse primers using the Bio-Rad iQ5 Quantitative PCR System (Takara, Dalian, China). The specific primers for TLR4 were sense, 5 -TTCAGAGCCGTTGGTGTATC-3 and antisense, 5 -CCCATTCCAGGTAGGTGTTT-3 ; for inducible NO synthase (iNOS) were sense, 5 -GGGAATCTTGGAGCGAGTTG-3 , and antisense, 5 -GTGAGGGCTTGGCTGAGTGA-3 ; and for -actin were sense, 5 -GATCATTGCTCCTCCTGAGC-3 and antisense, 5 ACTCCTGCTTGCTGATCCAC-3 . PCR were subjected to one cycle of 94 ◦ C for 10 min and then 40 cycles of 94 ◦ C for 30 s, 59 ◦ C for 30 and 72 ◦ C for 30 s. Gene expression changes were calculated by the comparative Ct method and the values were normalized to the control -actin. 2.8. Western blotting Total protein extracts were prepared using RIPA lysis buffer (Beyotime, Nantong, China) according to the operating instructions. The protein concentration in the lysates was evaluated using a BCA protein assay kit (Beyotime, Nantong, China). Equal amounts protein (30 g/lane) were separated on 10% SDS-PAGE and transferred onto polybinylidene difluoride membranes (Whatman Schleicher & Schuell, Middlesex, UK). The membranes were blocked in 5% (w/v) skimmed milk and then incubated with primary antibodies against mouse TLR4, phosphor-p65, I B␣ and -actin at 4 ◦ C overnight. Then the blots were washed three times
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Fig. 2. Effects of LRGP3 on macrophage viability. Cell viability was measured by MTT assay. LRGP3 did not affect cell viability at a concentration of 10 g/mL to 80 g/mL regardless of the presence of LPS. The values were presented as mean ± SD of three independent experiments. “−” means “RAW264.7 cells were not treated with LPS (1 g/mL) or LRGP3”; and “+” means “RAW264.7 cells were treated with LPS (1 g/mL)”.
with PBS containing 0.1% (w/v) Tween-20 and incubated with horseradish peroxidase-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA). Blots were again washed, and then developed by enhanced chemiluminescence detection reagent (Boehringer Mannheim, Mannheim, Germany). The protein band were quantified by the average ratios of integral optic density following normalization to the -actin. 2.9. Statistical analysis Statistical analyses were carried out with SPSS 13.0 software, and data were presented as mean ± SD. Statistical comparisons between multiple groups were performed using one-way ANOVA, followed by Dunnet’s post-hoc test. P < 0.05 was considered statistically significant.
3. Results 3.1. Effects of LRGP3 on macrophage viability Cell viability of mouse macrophage treated with a series of concentrations of LRGP3 and 1 g/mL LPS for 24 h were examined by the MTT method. As shown in Fig. 2, LRGP3 did not affect cell viability at a concentration of 10 g/mL to 80 g/mL regardless of the presence of LPS.
3.2. Effects of LRGP3 on NO production and iNOS mRNA expression in LPS-stimulated macrophages The release of NO from macrophage RAW264.7 cells stimulated by LRGP3 or LPS was detected. As shown in Fig. 3A, NO production was remarkably induced in LPS-induced RAW264.7 cells, as compared with un-stimulated negative control, while pretreatment with LRGP3 significantly prevented this increase in a dose-dependent manner. Furthermore, we investigated whether the inhibition of LRGP3 on NO production was related to downregulation of iNOS. As shown in Fig. 3B, the mRNA level of iNOS was significantly up-regulated in response to LPS, while treatment with LRGP3 could inhibit the mRNA expression of iNOS in a dosedependent manner. These results demonstrated that LRGP3 was able to inhibit the mRNA expression of iNOS, which in turn reduce the production of NO.
Fig. 3. Inhibition of LRGP3 of LPS-stimulated NO production and the level of iNOS mRNA. RAW264.7 cells were incubated for 24 h with the indicated concentrations of LRGP3 in the presence or absence of 1 g/mL LPS. (A) NO production was determined by Griess method; (B) mRNA level of iNOS was quantified by real-time PCR. Data are mean ± SD from three independent experiments performed in duplicate. # P < 0.01 compared with control group; * P < 0.05 compared with LPS group. “−” means “RAW264.7 cells were not treated with LPS (1 g/mL) or LRGP3”; and “+” means “RAW264.7 cells were treated with LPS (1 g/mL)”.
3.3. Suppression of LPS-stimulated TLR4 expression by LRGP3 in macrophages It has been reported that the TLR4 signaling pathway plays an important role in the development of inflammation. Therefore, we investigate the effects of LRGP3 on TLR4 expression in LPSstimulated macrophage. Western blot analysis showed that the level of TLR4 protein was decreased by treatment with LRGP3, as compared with the LPS-stimulated group (Fig. 4A). Moreover, consistent with the protein level, pretreatment of cells with the LRGP3 down-regulated the mRNA level of TLR4 (Fig. 4B). These data suggested that LRGP3 might provide protection from LPS-induced inflammation through suppression of TLR signaling pathway. 3.4. Suppression of LPS-stimulated I B˛ degradation and NF- B p65 phosphorylation by LRGP3 in macrophages Nuclear factor-kappa-binding (NF- B) is considered as a master switch in the regulation of inflammation and immunity. As a transcription factor, NF- B controls an array of pro-inflammatory genes involved in the inflammatory signaling cascade [10]. Therefore, we speculated that the anti-inflammatory effect of LRGP3 in response to LPS-induced inflammation correlated with blockade of NF- B activation. As shown in Fig. 5A, RAW264.7 cells treated with LPS exhibited significant degradation of inhibitor of B␣ (I B␣), whereas LRGP3 treatment prevented the I B␣ degradation in LPS-stimulated macrophages. In addition, up-regulation of phosphor-NF- B p65 was observed in LPS-stimulated mouse
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Fig. 4. Effect of LRGP3 on LPS-stimulated TLR4 expression. RAW264.7 cells were treated with medium only, LPS (1 g/mL), or LPS in the presence of increasing concentrations of LRGP3 (10–80 g/mL). (A) representative western blot of TLR4 after 24 h of treatment. The expression levels of proteins were normalized based on the -actin levels; (B) mRNA level of TLR4 was quantified by real-time PCR. Data are mean ± SD from three independent experiments performed in duplicate. # P < 0.01 compared with control group; * P < 0.05 compared with LPS group. “−” means “RAW264.7 cells were not treated with LPS (1 g/mL) or LRGP3”; and “+” means “RAW264.7 cells were treated with LPS (1 g/mL)”.
macrophages RAW264.7 cells, however, treatment with LRGP3 reduced phosphor-p65 expression in LPS-stimulated macrophages (Fig. 5B). These results suggested that LRGP3 significantly blocked the NF- B signaling pathway in LPS-stimulated macrophages by suppressing of I B␣ degradation and the phosphorylation of NF- B p65. 3.5. Suppression of pro-inflammatory cytokines by LRGP3 in LPS-stimulated macrophages To further determine the effect of LRP3 on NF- B signaling pathway, we investigated the expression levels of representative downstream signaling genes involved in NF- B activation. The releases of interleukin (IL)-1␣, IL-6 and TNF-␣ from RAW264.7 macrophages induced by LRGP3 or LPS were detected. As shown in Fig. 6, stimulation of the cells with LPS for 24 h increased the levels of IL-1␣, IL-6 and TNF-␣ in a concentration-dependent manner. In contrast, treatment of RAW264.7 cells with LRGP3 significantly attenuated the ability of LPS to increase the levels of cytokines. These data suggested that amelioration of inflammation in LPSstimulated RAW264.7 cells by LRP3 treatment correlated with repression of pro-inflammatory cytokines. 4. Discussion It has been reported that polysaccharides from natural sources are very potent anti-inflammatory drugs [11–13]. In this study, we demonstrated that LRGP3 could inhibit LPS-induced the production of NO and inflammatory cytokines. Moreover, we also documented the anti-inflammation mechanism of LRGP3 in LPSinduced macrophages.
Fig. 5. Effect of LRGP3 on LPS-stimulated activation of factors in the TLR4 signaling pathway. RAW264.7 cells were treated with medium only, LPS (1 g/mL), or LPS in the presence of increasing concentrations of LRGP3 (10–80 g/mL). (A) representative western blot of I B␣; (B) phosphorylation of NF- B p65 after 15 min of treatment was evaluated by western blot analysis. Data are mean ± SD from three independent experiments performed in duplicate. # P < 0.01 compared with control group; * P < 0.05 compared with LPS group. “−” means “RAW264.7 cells were not treated with LPS (1 g/mL) or LRGP3”; and “+” means “RAW264.7 cells were treated with LPS (1 g/mL)”.
LPS, the major component of the outer membrane of Gramnegative bacteria, is considered one of the main virulence factors of inflammation. The LPS injection model has been shown to induce inflammation in various cells by activating innate and adaptive immune responses, which modulate the expression of various inflammatory mediators [14,15]. In this study, we used the LPS-stimulated macrophage model to investigate the antiinflammatory effects of LRGP3. NO is highly reactive free radical involved in a large number of physiological and pathological processes in the inflammatory reaction [16]. It has been reported that NF- B activation mediates transactivation of iNOS [17], and NF- B sites identified in the iNOS gene promoter region, which activated by LPS [18]. In this study, we observed that pretreatment with LRGP3 significantly inhibited LPSinduced production of NO and the expression of iNOS in RAW264.7 cells, which suggested that the anti-inflammatory effects of LRGP3
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was 9253 pg/mL when 10 g/mL Bupleurum polysaccharide was applied to LPS-stimulated RAW264.7 cells, however, 10 g/mL LRGP3 decreased the content (8647 pg/mL). Therefore, we conclude that LRGP3 may play an important role in attenuating LPS-induced inflammation. TLR are type I transmembrane glycoproteins that recognize pathogen-associated and damage-associated molecular patterns. It has been reported that LPS could modulate TLR4 signaling pathway [23,24]. Our results found that LRGP3 inhibited the expression of TLR4 in LPS-stimulated macrophage. Polysaccharides and/or glycoproteins usually bind to surface receptors (e.g., TLR4, CD14, complement receptor 3, scavenger receptor, dectin-1 and mannose receptor) in macrophages and induce similar inflammatory responses by subsequent activation of intracellular signaling cascades, resulting in transcriptional activation and production of inflammatory cytokines [25]. Wu et al. reported that Bupleurum polysaccharides ameliorated lung injuries and suppressed TLR expression in a rat model of acute lung injury with pulmonary hemorrhage and inflammation [26]. NF B signaling is considered a pivotal mechanism for the regulation of immune and inflammatory responses by controlling the transcription of inflammatory cytokine genes [27]. Activation of NF- B involves in the phosphorylation and subsequent proteolytic degradation of the inhibitory protein I B kinases [28]. Our results showed that LPS caused the phosphorylation of NF- B p65 and I B␣ degradation in mouse macrophages RAW264.7 cells, while treatment with LRGP3 reduced the expression of phosphor-p65 and prevented I B␣ degradation in LPS-stimulated macrophages, suggest that LRGP3 might inhibit NF- B activation due to its inhibition of I B␣ degradation and phosphorylation of NF- B p65. In conclusion, LRGP3 suppressed LPS-induced inflammation via modulating TLR4/NF- B signaling pathway in RAW264.7 cells. These results suggest that LRGP3 could represent a potential antiinflammatory drug and this new beneficial effect may expand future researchers on anti-inflammatory properties of LRGP3 in vivo. Acknowledgment This research was financially supported by the doctoral research fund of Northwest A&F University (2013BSJJ079). References Fig. 6. Effects of LRGP3 on LPS-stimulated inflammatory cytokines. RAW264.7 cells were treated with medium only, LPS (1 g/mL), or LPS in the presence of increasing concentrations of LRGP3 (10–80 g/mL). Supernatants were analyzed for (A) IL-␣, (B) IL-6, (C) TNF-␣. Data are mean ± SD from three independent experiments performed in duplicate. # P < 0.01 compared with control group; * P < 0.05 compared with LPS group. “−” means “RAW264.7 cells were not treated with LPS (1 g/mL) or LRGP3”; and “+” means “RAW264.7 cells were treated with LPS (1 g/mL)”.
may be mediated, at least in part, by the NF- B-iNOS-NO signaling pathway. During infections, the pro-inflammatory cytokines act first in the inflammation process [19]. TNF-␣ is a major mediator in inflammatory responses and can induce innate immune responses by activating macrophages and by stimulating secretion of other inflammatory cytokines [20]. Recent studies indicated that a lotus plumule polysaccharide has strong anti-inflammatory effects on LPS-induced inflamed macrophages in preventive manner [21]; treatment with Bupleurum polysaccharides enhanced phagocytic functions of macrophages and inhibited LPS-induced productions of pro-inflammatory cytokines [22]. Consistent with other reports, in this study, we found that LRGP3 significantly decrease LPSinduced cytokines levels in RAW264.7 cells. The content of TNF-␣
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Lycium ruthenicum polysaccharide attenuates inflammation through inhibiting TLR4/NF- B signaling pathway Qiang Peng a,∗ , Huajing Liu b , Shihui Shi a , Ming Li c,∗ a b c
College of Food Science and Engineering, Northwest A&F University, Yangling 712100, China College of Resources and Environmental Science, Northeast Agricultural University, Harbin 150030, China College of Resources and Environment, Northwest A&F University, YangLing 712100, China
a r t i c l e
i n f o
Article history: Received 12 January 2014 Received in revised form 7 March 2014 Accepted 8 March 2014 Available online 25 March 2014 Keywords: Lycium ruthenicum Polysaccharide Toll-like receptor 4 NF- B
a b s t r a c t Polysaccharide has been reported to possess diverse biological activities, however, the inflammatory activity of polysaccharide isolated from Lycium ruthenicum remains unknown so far. In the present study, we investigated the effects of L. ruthenicum polysaccharide (LRGP3) on inflammatory reaction induced by lipopolysaccharide (LPS) in mouse macrophage RAW264.7 cells and some potential underlying mechanisms. Our results showed that LRGP3 treatment significantly inhibited the LPS-induced NO production and the mRNA expression of iNOS, as well as the level of Toll-like receptor 4 (TLR4). Furthermore, LRGP3 treatment prevented the I B␣ degradation and reduced phospho-NF- B p65 protein expression in LPSstimulated RAW264.7 cells. Meanwhile, the levels of pro-inflammatory cytokines, such as interleukin (IL)-␣, IL-6, tumor necrosis factor (TNF)-␣ were suppressed by LRGP3 in LPS-stimulated RAW264.7 cells. Taken together, our results suggested that LRGP3 attenuated LPS-induced inflammation via inhibiting TLR4/NF- B signaling pathway. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Inflammation, a physiological response to infection or injury, plays an important in various diseases, such as atherosclerosis, neurodegenerative disease, cancers. During the process of inflammation, different cell types are recruited, including monocytes that differentiate locally into macrophages. This leads to the regulated production of various pro- and antiinflammatory mediators including cytokines, such as interleukins (ILs), interferon, tumor necrosis factor (TNF)-␣ [1,2]. Previous studies showed that traditional Chinese medicine herbs possess anti-inflammatory property. Forsythiaside, the major bioactive component of Forsythia suspense Vahl., has been shown to exhibit a promising anti-inflammatory activity by decreasing the expression of pro-inflammatory cytokines in lipopolysaccharide (LPS)-induced inflammation on the bursa of Fabricius of chickens [3]. Ethanol extract from a traditional Chinese medicinal decoction,
∗ Corresponding authors at: Northwest A&F University, College of Food Science and Engineering, Xinong Road 22, Yangling, China. Tel.: +86 29 87091746/+86 451 87091793; fax: +86 29 87091746/+86 451 55190720. E-mail addresses: [email protected], [email protected] (Q. Peng), [email protected] (M. Li). http://dx.doi.org/10.1016/j.ijbiomac.2014.03.023 0141-8130/© 2014 Elsevier B.V. All rights reserved.
“Zuojin Pill”, inhibited the expression of inflammatory mediators in LPS-stimulated RAW264.7 mouse macrophages [4]. The phenolic glucoside, a main constituent of a Chinese herbal medicine, attenuated levels of neurotoxic proinflammatory mediators and proinflammatory cytokines in LPS-stimulated microglial cells [5]. Ginsenoside Rg1 also attenuated overactivation of microglial cells by repressing expression levels of neurotoxic proinflammatory cytokines in LPS-stimulated murine microglial cells [6]. Lycium ruthenicum Murr., belongs to the genus Lycium of the family Solanaceae, and is a native wild resource plant of China, found principally in Qinghai and Xinjiang Provinces. Active constituents of L. ruthenicum Murr. are reported to be have a variety of biological activities, including immunoregulation, anti-aging, lowering blood-sugar and blood-fat levels and anti-fatigue. Of these ingredients, polysaccharide has been known as the major bioactive compound. However, purified polysaccharide with antiinflammatory activity has not yet been documented. Recently, we have purified a water-soluble polysaccharide (LRGP3) from the crude polysaccharide extracted from L. ruthenicum Murr, and the structure of LRGP3 was shown in Fig. 1. LRGP3 was a highly branched polysaccharide with a backbone of (1 → 3)-linked -d-galactopyranosyl residues, many of which were substituted at the O-6 position. The branches were composed of (1 → 5)-linked arabinosyl, (1 → 2)-linked arabinosyl,
Q. Peng et al. / International Journal of Biological Macromolecules 67 (2014) 330–335
331
Fig. 1. The structure of the repeat unit of the glycan of LRGP3.
(1 → 6)-linked galactosyl, (1 → 3)-linked galactosyl, and (1 → 2,4)linked rhamnosyl residues, and the major nonreducing termini were ␣-l-arabinofuranosyl residues [7]. In the present study, we investigated the anti-inflammatory activity of LRGP3 in LPS-stimulated mouse macrophage RAW264.7 cells by examining changes in levels of NO production and pro-inflammatory cytokines. Furthermore, the expression of Toll-like receptor 4 (TLR4) and downstream molecules of TLR4 signaling pathway were examined to investigate the potential mechanisms involved in the effects of LRGP3.
was recorded using a PR 4100 microplate reader (Bio-Rad, Bio-Rad, Hercules, CA, USA).
2. Materials and methods
Quantitative determination in the cell culture medium was made using the Enzyme-Linked Immunosorbent Assay (ELISA) kit (Invitrogen, Carlsbad, CA, USA) for IL-1␣, IL-6 and TNF-␣, according to manufacturer’s instruction, and the results are expressed in pg/mg of protein in each sample. All of the analyses were performed in triplicate.
2.1. Materials The anti-TLR4 antibody, anti-phospho-p65, anti-I B␣ and anti -actin antibodies were purchased from Invitrogen (Carlsbad, CA). All other chemicals and reagents were purchased from Sigma (Saint Louis, MO). 2.2. Preparation of LRGP3 The L. ruthenicum Murr. was extracted twice with boiling water for 2 h at a ratio of 20:1 (w/w) followed by centrifuging at 6000 rpm × 10 min. Supernatants were combined and concentrated to 1/5 of the original volume, and precipitated by adding 95% ethanol (4 vol). After centrifugation, the precipitate was dissolved with 500 mL of water and subjected to Savage method to remove free protein [8]. The de-proteinization solution was concentrated, dialyzed against distilled water, and lyophilized to obtain crude polysaccharides. The purity of CLRP was performed as previously described [7]. 2.3. Cell culture RAW264.7 mouse macrophage cells were obtained from American Type Cultured Collection (Rockville, MD, USA) and cultured in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum, glutamine, and antibiotics at 37 ◦ C under 5% CO2 . 2.4. Cell viability assay The viability of cells was measured using MTT assay. In brief, 5 × 103 cells/well RAW264.7 cells were plated in 96-well microplates overnight and then treated with different concentrations of LRGP3 or LPS (1 g/mL) for 24 h. Equal volume of medium was used as vehicle control. After treatment, cells were incubated with 0.5 mg/mL of MTT for another 4 h in dark and then the medium was discarded. The formazan crystals presented in cells were dissolved by 100 L dimethyl sulfoxide. The absorbance at 570 nm
2.5. NO production After preincubation of RAW264.7 cells for 18 h, cells were treated with LRGP3 (10–80 g/mL) or LPS (1 g/mL) for 24 h. Griess reagent was used to determine NO production [9]. 2.6. Quantitative analysis of cytokines
2.7. Real time-PCR Total RNA was extracted from RAW264.7 cells using TRIzol Reagent (Invitrogen). And mRNA was reversed transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Equal amounts of cDNA were submitted for PCR in the presence of SYBR Green I reagent and forward and reverse primers using the Bio-Rad iQ5 Quantitative PCR System (Takara, Dalian, China). The specific primers for TLR4 were sense, 5 -TTCAGAGCCGTTGGTGTATC-3 and antisense, 5 -CCCATTCCAGGTAGGTGTTT-3 ; for inducible NO synthase (iNOS) were sense, 5 -GGGAATCTTGGAGCGAGTTG-3 , and antisense, 5 -GTGAGGGCTTGGCTGAGTGA-3 ; and for -actin were sense, 5 -GATCATTGCTCCTCCTGAGC-3 and antisense, 5 ACTCCTGCTTGCTGATCCAC-3 . PCR were subjected to one cycle of 94 ◦ C for 10 min and then 40 cycles of 94 ◦ C for 30 s, 59 ◦ C for 30 and 72 ◦ C for 30 s. Gene expression changes were calculated by the comparative Ct method and the values were normalized to the control -actin. 2.8. Western blotting Total protein extracts were prepared using RIPA lysis buffer (Beyotime, Nantong, China) according to the operating instructions. The protein concentration in the lysates was evaluated using a BCA protein assay kit (Beyotime, Nantong, China). Equal amounts protein (30 g/lane) were separated on 10% SDS-PAGE and transferred onto polybinylidene difluoride membranes (Whatman Schleicher & Schuell, Middlesex, UK). The membranes were blocked in 5% (w/v) skimmed milk and then incubated with primary antibodies against mouse TLR4, phosphor-p65, I B␣ and -actin at 4 ◦ C overnight. Then the blots were washed three times
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Fig. 2. Effects of LRGP3 on macrophage viability. Cell viability was measured by MTT assay. LRGP3 did not affect cell viability at a concentration of 10 g/mL to 80 g/mL regardless of the presence of LPS. The values were presented as mean ± SD of three independent experiments. “−” means “RAW264.7 cells were not treated with LPS (1 g/mL) or LRGP3”; and “+” means “RAW264.7 cells were treated with LPS (1 g/mL)”.
with PBS containing 0.1% (w/v) Tween-20 and incubated with horseradish peroxidase-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA). Blots were again washed, and then developed by enhanced chemiluminescence detection reagent (Boehringer Mannheim, Mannheim, Germany). The protein band were quantified by the average ratios of integral optic density following normalization to the -actin. 2.9. Statistical analysis Statistical analyses were carried out with SPSS 13.0 software, and data were presented as mean ± SD. Statistical comparisons between multiple groups were performed using one-way ANOVA, followed by Dunnet’s post-hoc test. P < 0.05 was considered statistically significant.
3. Results 3.1. Effects of LRGP3 on macrophage viability Cell viability of mouse macrophage treated with a series of concentrations of LRGP3 and 1 g/mL LPS for 24 h were examined by the MTT method. As shown in Fig. 2, LRGP3 did not affect cell viability at a concentration of 10 g/mL to 80 g/mL regardless of the presence of LPS.
3.2. Effects of LRGP3 on NO production and iNOS mRNA expression in LPS-stimulated macrophages The release of NO from macrophage RAW264.7 cells stimulated by LRGP3 or LPS was detected. As shown in Fig. 3A, NO production was remarkably induced in LPS-induced RAW264.7 cells, as compared with un-stimulated negative control, while pretreatment with LRGP3 significantly prevented this increase in a dose-dependent manner. Furthermore, we investigated whether the inhibition of LRGP3 on NO production was related to downregulation of iNOS. As shown in Fig. 3B, the mRNA level of iNOS was significantly up-regulated in response to LPS, while treatment with LRGP3 could inhibit the mRNA expression of iNOS in a dosedependent manner. These results demonstrated that LRGP3 was able to inhibit the mRNA expression of iNOS, which in turn reduce the production of NO.
Fig. 3. Inhibition of LRGP3 of LPS-stimulated NO production and the level of iNOS mRNA. RAW264.7 cells were incubated for 24 h with the indicated concentrations of LRGP3 in the presence or absence of 1 g/mL LPS. (A) NO production was determined by Griess method; (B) mRNA level of iNOS was quantified by real-time PCR. Data are mean ± SD from three independent experiments performed in duplicate. # P < 0.01 compared with control group; * P < 0.05 compared with LPS group. “−” means “RAW264.7 cells were not treated with LPS (1 g/mL) or LRGP3”; and “+” means “RAW264.7 cells were treated with LPS (1 g/mL)”.
3.3. Suppression of LPS-stimulated TLR4 expression by LRGP3 in macrophages It has been reported that the TLR4 signaling pathway plays an important role in the development of inflammation. Therefore, we investigate the effects of LRGP3 on TLR4 expression in LPSstimulated macrophage. Western blot analysis showed that the level of TLR4 protein was decreased by treatment with LRGP3, as compared with the LPS-stimulated group (Fig. 4A). Moreover, consistent with the protein level, pretreatment of cells with the LRGP3 down-regulated the mRNA level of TLR4 (Fig. 4B). These data suggested that LRGP3 might provide protection from LPS-induced inflammation through suppression of TLR signaling pathway. 3.4. Suppression of LPS-stimulated I B˛ degradation and NF- B p65 phosphorylation by LRGP3 in macrophages Nuclear factor-kappa-binding (NF- B) is considered as a master switch in the regulation of inflammation and immunity. As a transcription factor, NF- B controls an array of pro-inflammatory genes involved in the inflammatory signaling cascade [10]. Therefore, we speculated that the anti-inflammatory effect of LRGP3 in response to LPS-induced inflammation correlated with blockade of NF- B activation. As shown in Fig. 5A, RAW264.7 cells treated with LPS exhibited significant degradation of inhibitor of B␣ (I B␣), whereas LRGP3 treatment prevented the I B␣ degradation in LPS-stimulated macrophages. In addition, up-regulation of phosphor-NF- B p65 was observed in LPS-stimulated mouse
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Fig. 4. Effect of LRGP3 on LPS-stimulated TLR4 expression. RAW264.7 cells were treated with medium only, LPS (1 g/mL), or LPS in the presence of increasing concentrations of LRGP3 (10–80 g/mL). (A) representative western blot of TLR4 after 24 h of treatment. The expression levels of proteins were normalized based on the -actin levels; (B) mRNA level of TLR4 was quantified by real-time PCR. Data are mean ± SD from three independent experiments performed in duplicate. # P < 0.01 compared with control group; * P < 0.05 compared with LPS group. “−” means “RAW264.7 cells were not treated with LPS (1 g/mL) or LRGP3”; and “+” means “RAW264.7 cells were treated with LPS (1 g/mL)”.
macrophages RAW264.7 cells, however, treatment with LRGP3 reduced phosphor-p65 expression in LPS-stimulated macrophages (Fig. 5B). These results suggested that LRGP3 significantly blocked the NF- B signaling pathway in LPS-stimulated macrophages by suppressing of I B␣ degradation and the phosphorylation of NF- B p65. 3.5. Suppression of pro-inflammatory cytokines by LRGP3 in LPS-stimulated macrophages To further determine the effect of LRP3 on NF- B signaling pathway, we investigated the expression levels of representative downstream signaling genes involved in NF- B activation. The releases of interleukin (IL)-1␣, IL-6 and TNF-␣ from RAW264.7 macrophages induced by LRGP3 or LPS were detected. As shown in Fig. 6, stimulation of the cells with LPS for 24 h increased the levels of IL-1␣, IL-6 and TNF-␣ in a concentration-dependent manner. In contrast, treatment of RAW264.7 cells with LRGP3 significantly attenuated the ability of LPS to increase the levels of cytokines. These data suggested that amelioration of inflammation in LPSstimulated RAW264.7 cells by LRP3 treatment correlated with repression of pro-inflammatory cytokines. 4. Discussion It has been reported that polysaccharides from natural sources are very potent anti-inflammatory drugs [11–13]. In this study, we demonstrated that LRGP3 could inhibit LPS-induced the production of NO and inflammatory cytokines. Moreover, we also documented the anti-inflammation mechanism of LRGP3 in LPSinduced macrophages.
Fig. 5. Effect of LRGP3 on LPS-stimulated activation of factors in the TLR4 signaling pathway. RAW264.7 cells were treated with medium only, LPS (1 g/mL), or LPS in the presence of increasing concentrations of LRGP3 (10–80 g/mL). (A) representative western blot of I B␣; (B) phosphorylation of NF- B p65 after 15 min of treatment was evaluated by western blot analysis. Data are mean ± SD from three independent experiments performed in duplicate. # P < 0.01 compared with control group; * P < 0.05 compared with LPS group. “−” means “RAW264.7 cells were not treated with LPS (1 g/mL) or LRGP3”; and “+” means “RAW264.7 cells were treated with LPS (1 g/mL)”.
LPS, the major component of the outer membrane of Gramnegative bacteria, is considered one of the main virulence factors of inflammation. The LPS injection model has been shown to induce inflammation in various cells by activating innate and adaptive immune responses, which modulate the expression of various inflammatory mediators [14,15]. In this study, we used the LPS-stimulated macrophage model to investigate the antiinflammatory effects of LRGP3. NO is highly reactive free radical involved in a large number of physiological and pathological processes in the inflammatory reaction [16]. It has been reported that NF- B activation mediates transactivation of iNOS [17], and NF- B sites identified in the iNOS gene promoter region, which activated by LPS [18]. In this study, we observed that pretreatment with LRGP3 significantly inhibited LPSinduced production of NO and the expression of iNOS in RAW264.7 cells, which suggested that the anti-inflammatory effects of LRGP3
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was 9253 pg/mL when 10 g/mL Bupleurum polysaccharide was applied to LPS-stimulated RAW264.7 cells, however, 10 g/mL LRGP3 decreased the content (8647 pg/mL). Therefore, we conclude that LRGP3 may play an important role in attenuating LPS-induced inflammation. TLR are type I transmembrane glycoproteins that recognize pathogen-associated and damage-associated molecular patterns. It has been reported that LPS could modulate TLR4 signaling pathway [23,24]. Our results found that LRGP3 inhibited the expression of TLR4 in LPS-stimulated macrophage. Polysaccharides and/or glycoproteins usually bind to surface receptors (e.g., TLR4, CD14, complement receptor 3, scavenger receptor, dectin-1 and mannose receptor) in macrophages and induce similar inflammatory responses by subsequent activation of intracellular signaling cascades, resulting in transcriptional activation and production of inflammatory cytokines [25]. Wu et al. reported that Bupleurum polysaccharides ameliorated lung injuries and suppressed TLR expression in a rat model of acute lung injury with pulmonary hemorrhage and inflammation [26]. NF B signaling is considered a pivotal mechanism for the regulation of immune and inflammatory responses by controlling the transcription of inflammatory cytokine genes [27]. Activation of NF- B involves in the phosphorylation and subsequent proteolytic degradation of the inhibitory protein I B kinases [28]. Our results showed that LPS caused the phosphorylation of NF- B p65 and I B␣ degradation in mouse macrophages RAW264.7 cells, while treatment with LRGP3 reduced the expression of phosphor-p65 and prevented I B␣ degradation in LPS-stimulated macrophages, suggest that LRGP3 might inhibit NF- B activation due to its inhibition of I B␣ degradation and phosphorylation of NF- B p65. In conclusion, LRGP3 suppressed LPS-induced inflammation via modulating TLR4/NF- B signaling pathway in RAW264.7 cells. These results suggest that LRGP3 could represent a potential antiinflammatory drug and this new beneficial effect may expand future researchers on anti-inflammatory properties of LRGP3 in vivo. Acknowledgment This research was financially supported by the doctoral research fund of Northwest A&F University (2013BSJJ079). References Fig. 6. Effects of LRGP3 on LPS-stimulated inflammatory cytokines. RAW264.7 cells were treated with medium only, LPS (1 g/mL), or LPS in the presence of increasing concentrations of LRGP3 (10–80 g/mL). Supernatants were analyzed for (A) IL-␣, (B) IL-6, (C) TNF-␣. Data are mean ± SD from three independent experiments performed in duplicate. # P < 0.01 compared with control group; * P < 0.05 compared with LPS group. “−” means “RAW264.7 cells were not treated with LPS (1 g/mL) or LRGP3”; and “+” means “RAW264.7 cells were treated with LPS (1 g/mL)”.
may be mediated, at least in part, by the NF- B-iNOS-NO signaling pathway. During infections, the pro-inflammatory cytokines act first in the inflammation process [19]. TNF-␣ is a major mediator in inflammatory responses and can induce innate immune responses by activating macrophages and by stimulating secretion of other inflammatory cytokines [20]. Recent studies indicated that a lotus plumule polysaccharide has strong anti-inflammatory effects on LPS-induced inflamed macrophages in preventive manner [21]; treatment with Bupleurum polysaccharides enhanced phagocytic functions of macrophages and inhibited LPS-induced productions of pro-inflammatory cytokines [22]. Consistent with other reports, in this study, we found that LRGP3 significantly decrease LPSinduced cytokines levels in RAW264.7 cells. The content of TNF-␣
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