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Inhibition of IL-6 and IL-8 production in LPS-stimulated human gingival fibroblasts by glycyrrhizin via activating LXRα
Microbial Pathogenesis 110 (2017) 135e139
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Inhibition of IL-6 and IL-8 production in LPS-stimulated human gingival fibroblasts by glycyrrhizin via activating LXRa Na Zhang a, b, Hui Lv b, Bo-Hong Shi b, Xin Hou b, Xin Xu a, * a b
Shandong Provincial Key Laboratory of Oral Tissue Regeneration, School of Stomatology, Shandong University, Shandong, China Department of Stomatology, The People's Hospital of Rizhao, Shandong, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 May 2017 Received in revised form 8 June 2017 Accepted 15 June 2017 Available online 23 June 2017
The aim of this study was to clarify the anti-inflammatory effects and its molecular mechanism of glycyrrhizin on LPS-stimulated human gingival fibroblasts (HGFs), which will be of benefit for periodontitis treatment. An MTT assay was performed to assess the effects of glycyrrhizin on cellular viability. The levels of IL-6 and IL-8 were measured by ELISA. The expression of iNOS, COX-2, NF-kB, and LXRa were detected by western blot analysis. The results showed that glycyrrhizin significantly inhibited LPSinduced IL-6 and IL-8 production, as well as COX-2 and iNOS expression. LPS-induced NF-kB activation in HGFs was also inhibited by treatment of glycyrrhizin. Furthermore, glycyrrhizin increased the expression of LXRa in a concentration-dependent manner. In addition, the inhibition of glycyrrhizin on IL-6 and IL-8 production was reversed by LXRa inhibitor GGPP. In conclusion, these results indicated that glycyrrhizin exhibited its anti-inflammatory effects in HGFs by activating LXRa. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Glycyrrhizin Human gingival fibroblasts IL-8 NF-kB LXRa
1. Introduction Periodontitis is a common and often undiagnosed oral disease that characterized by inflammatory destruction of periodontal tissues [1]. Periodontal destruction is often caused by bacteria, which leads to the inflammation and bone resorption [2]. Porphyromonas gingivalis is the major pathogen that leads to periodontitis [3,4]. Porphyromonas gingivalis LPS could induce NF-kB activation and inflammatory mediators release in human gingival fibroblasts [5]. These inflammatory mediators, such as IL-6, IL-8, NO, and PGE2, have the ability to induce periodontal tissue injury [6]. Previous studies showed that inhibition of inflammatory response could attenuate the pathological process of periodontitis [7]. LXRa is a nuclear receptor that has previously been shown to regulate inflammatory response [8]. It has been reported that activating LXRa could inhibit LPS-induced NF-kB activation [9]. Therefore, LXRa may be used as a target for the treatment of periodontitis. Glycyrrhizin, an active component of liquorice roots, has been reported to have anti-inflammatory effect. Previous study showed that glycyrrhizin inhibited LPS and D-galactosamine-induced liver injury in mice [10]. Glycyrrhizin also suppressed LPS-induced acute
* Corresponding author. E-mail address: [email protected] (X. Xu). http://dx.doi.org/10.1016/j.micpath.2017.06.021 0882-4010/© 2017 Elsevier Ltd. All rights reserved.
lung injury in mice by inhibiting inflammatory mediators production [11]. Glycyrrhizin was found to inhibit LPS-induced inflammatory cytokines production in mammary epithelial cells [12]. Moreover, glycyrrhizin has been known to inhibit HMGB1 secretion in LPS-stimulated RAW264.7 cells [13]. LPS-induced neuroinflammation and memory deficit were also inhibited by treatment of glycyrrhizin [14]. However, the anti-inflammatory effects of glycyrrhizin on LPS-induced inflammatory response in HGFs have not been reported. The aim of this study was to clarify the antiinflammatory effects and mechanism of glycyrrhizin in LPSstimulated HGFs. 2. Materials and methods 2.1. Chemicals and reagents Glycyrrhizin (purity>98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). GGPP was purchased from Sigma-Aldrich (CA, USA). LPS from Porphyromonas gingivalis was obtained from InvivoGen (San Diego, CA, USA). NF-kB p65, NF-kB p-p65, IkBa, p-IkBa, iNOS, COX-2, and b-actin were purchased from Cell Signaling Technology Inc (Beverly, MA). LXRa antibody was purchased from Santa Cruz Biotechnology. ELISA kits for IL-6 and IL-8 were purchased from R&D. All other reagents were of analytical grade.
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2.2. Cell culture HGFs were isolated from explants of human normal gingival tissues as described previously [15]. This study was approved by the local ethic committee. The cells were suspended in DMEM containing 10% fetal bovine serum (FBS). Cells between passages 3 to 8 were used in this study. For LXRa inhibitory experiment, the cells were pretreated with or without 20 mM geranylgeranyl pyrophosphate (GGPP) for 2 h, then the cells were pretreated with glycyrrhizin 12 h before LPS treatment. 2.3. Cell viability Cell viability was detected by MTT assay. In brief, HGFs were seeded at a 96-well plate at the density of 1 104 cells/well. Then, the cells were treated with LPS and glycyrrhizin for 24 h. MTT was added to each well. 4 h later, 150 mL of DMSO was added to each well. Optical density was measured at 450 nm using a Bio-Rad Microplate Reader (Model 680, Bio-Rad, USA).
Fig. 1. Effects of glycyrrhizin on the cell viability of HGFs. Cells were cultured with different concentrations of glycyrrhizin (0e180 mg/ml) in the absence or presence of 1 mg/ml LPS for 24 h. The cell viability was determined by MTT assay. The values presented are the means ± SD of three independent experiments.
2.4. Cytokines assay The culture medium was collected and the production of cytokines IL-6 and IL-8 in the culture medium were measured using ELISA kits (R&D) according to the manufacturer's instructions. 2.5. Western blot analysis HGFs were lysed using RIPA lysis buffer and the protein concentration was detected by BCA method. The protein (30 mg) were separated by 12% SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad, PA, USA). The membranes were incubated with antibodies against COX-2, iNOS, NF-kB p65, NF-kB p-p65, IkBa, p-IkBa, LXRa, and b-actin overnight at 4 C. Then, the membranes were probed with HRP-conjugated secondary antibody for 1 h. The specific binding was detected using chemiluminescence detection system (Amersham, Berkshire, UK). 2.6. Statistical analysis Results were presented as means ± SD. Differences between multiple treatments were analyzed with one-way analysis followed by the Newman-Keuls post hoc test. Statistically significance were considered at P < 0.05. 3. Results 3.1. Effects of glycyrrhizin on HGFs cell viability Effects of glycyrrhizin on HGFs cell viability was assessed by the MTT assay. As shown in Fig. 1, glycyrrhizin at the doses of 40, 80, 160 mg/ml did not affect the cell viabilities of HGFs. Glycyrrhizin at the dose of 180 mg/ml decreased the cell viabilities of HGFs. Thus, in the subsequent studies, glycyrrhizin at the doses of 40, 80, 160 mg/ ml were used. 3.2. Glycyrrhizin inhibits LPS-induced IL-6 and IL-8 production in HGFs To evaluate the anti-inflammatory effects of glycyrrhizin on LPSstimulated HGFs, the production of IL-6 and IL-8 were detected. As shown in Fig. 2, LPS treatment increased the production of IL-6 and IL-8 compared with the untreated group. However, pre-treatment of the cells with glycyrrhizin decreased the production of IL-6 and IL-8 compared with the LPS group.
Fig. 2. Glycyrrhizin inhibits LPS-induced IL-8 and IL-6 production in HGFs. The data presented are the means ± SD of three independent experiments. #p < 0.05 vs. control group; *p < 0.05, **p < 0.01 vs. LPS group.
3.3. Glycyrrhizin inhibits LPS-induced COX-2 and iNOS expression in HGFs To evaluate the anti-inflammatory effects of glycyrrhizin on LPSstimulated HGFs, the production of IL-6 and IL-8 were detected. As shown in Fig. 3, LPS treatment increased the production of IL-6 and IL-8 compared with the untreated group. However, pre-treatment of the cells with glycyrrhizin decreased the production of IL-6 and IL-8 compared with the LPS group.
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Fig. 3. Effects of glycyrrhizin on iNOS and COX-2 expression in LPS-stimulated HGFs. The data presented are the means ± SD of three independent experiments. #p < 0.05 vs. control group; *p < 0.05, **p < 0.01 vs. LPS group.
3.4. Glycyrrhizin inhibits LPS-induced NF-kB activation in HGFs NF-kB is essential for the expression of inflammatory mediators. Thus, we investigated whether glycyrrhizin could inhibit LPSinduced NF-kB activation. As shown in Fig. 4, LPS treatment induced IkB-a phosphorylation and degradation and NF-kB p65 phosphorylation. However, treatment of glycyrrhizin significantly inhibited LPS-induced IkB-a phosphorylation and degradation and NF-kB p65 phosphorylation. 3.5. Glycyrrhizin exhibits anti-inflammatory effects in HGFs by activating LXRa To further investigate the anti-inflammatory mechanism of glycyrrhizin, the effects of glycyrrhizin on LXRa expression were
detected in this study. As shown in Fig. 5A, glycyrrhizin concentration-dependently up-regulated the expression of LXRa. Furthermore, our results showed that the inhibition of glycyrrhizin on IL-6 and IL-8 production can be reversed by LXRa inhibitor GGPP (Fig. 5B). 4. Discussion In the present study, we designed to detect whether glycyrrhizin could inhibit LPS-induced inflammatory response in HGFs, which has been associated with the development of periodontal diseases. We found that glycyrrhizin inhibited LPS-induced inflammatory mediators production by activating LXRa. Porphyromonas gingivalis is a major pathogen of periodontal disease that leads to inflammatory response in the gingival [16].
Fig. 4. Effects of glycyrrhizin on LPS-induced NF-kB activation and IkBa degradation. The values presented are the means ± SD of three independent experiments. #p < 0.05 vs. control group; *p < 0.05, **p < 0.01 vs. LPS group.
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effects in LPS-stimulated HGFs. LXRa is a member of the nuclear hormone receptor superfamily that has been reported to regulate the metabolism of lipids [20,21]. A large body of studies showed that LXR agonists possessed antiinflammatory properties [22,23]. Activation of LXRa could attenuate the activation of transcription factor NF-kB [24]. Studies showed that activation of NF-kB contributed to the pathologic process of periodontitis [25]. Stimulating of HGFs with LPS could induce the activation of NF-kB [26]. Previous studies showed that inhibition of NF-kB activation could attenuate the pathologic process of periodontitis [27]. In the present study, our results showed that glycyrrhizin significantly inhibited LPS-induced NF-kB activation. In addition, we found that glycyrrhizin concentrationdependently up-regulated the expression of LXRa. The antiinflammatory effects of glycyrrhizin were blocked by LXRa inhibitor GGPP. These results suggested that glycyrrhizin inhibited LPSinduced inflammatory response in HGFs by activating LXRa. In conclusion, our results found that glycyrrhizin significantly suppressed LPS-induced IL-6 and IL-8 production in HGFs. The antiinflammatory mechanism was through activating LXRa, which subsequently inhibited LPS-induced NF-kB activation. These results suggested that glycyrrhizin may provide a beneficial effect for treating inflammation and periodontal diseases. Conflict of interest All authors declare that they have no conflict of interest. References
Fig. 5. (A) Effects of glycyrrhizin on LXRa expression. (B) Block LXRa reversed the antiinflammatory effects of glycyrrhizin. The values presented are the means ± SD of three independent experiments. #P < 0.05 vs. control group; *P < 0.05, **P < 0.01 vs. LPS group.
Stimulation of gingival fibroblasts by Porphyromonas gingivalis LPS induced the release of inflammatory mediators [17]. These inflammatory mediators played critical roles in the development of periodontitis [1]. Previous studies showed that inhibition of these inflammatory mediators might attenuate the pathologic process of periodontitis [18,19]. To explore the anti-inflammatory effects of glycyrrhizin, the effects of glycyrrhizin on LPS-induced inflammatory mediators production were detected. Our results demonstrated that glycyrrhizin significantly inhibited LPS-induced IL-6 and IL-8 production, as well as COX-2 and iNOS expression. The results suggested that glycyrrhizin exhibited anti-inflammatory
[1] R.C. Page, The role of inflammatory mediators in the pathogenesis of periodontal disease, J. periodontal Res. 26 (1991) 230e242. [2] S.S. Socransky, Relationship of bacteria to the etiology of periodontal disease, J. Dent. Res. 49 (1970) 203e222. [3] H. Kuula, T. Salo, E. Pirila, A.M. Tuomainen, M. Jauhiainen, V.J. Uitto, et al., Local and systemic responses in matrix metalloproteinase 8-deficient mice during Porphyromonas gingivalis-induced periodontitis, Infect. Immun. 77 (2009) 850e859. [4] J. Mysak, S. Podzimek, P. Sommerova, Y. Lyuya-Mi, J. Bartova, T. Janatova, et al., Porphyromonas gingivalis: major periodontopathic pathogen overview, J. Immunol. Res. 2014 (2014) 476068. [5] T.D. Herath, R.P. Darveau, C.J. Seneviratne, C.Y. Wang, Y. Wang, L. Jin, Tetraand penta-acylated lipid A structures of Porphyromonas gingivalis LPS differentially activate TLR4-mediated NF-kappaB signal transduction cascade and immuno-inflammatory response in human gingival fibroblasts, PloS one 8 (2013) e58496. [6] A.I. Dongari-Bagtzoglou, J.L. Ebersole, Production of inflammatory mediators and cytokines by human gingival fibroblasts following bacterial challenge, J. periodontal Res. 31 (1996) 90e98. [7] T.E. Van Dyke, Resolution of inflammation-unraveling mechanistic links between periodontitis and cardiovascular disease, J. Dent. 37 (2009) S582eS583. [8] C. Hong, P. Tontonoz, Coordination of inflammation and metabolism by PPAR and LXR nuclear receptors, Curr. Opin. Genet. Dev. 18 (2008) 461e467. [9] S. Wu, R. Yin, R. Ernest, Y. Li, O. Zhelyabovska, J. Luo, et al., Liver X receptors are negative regulators of cardiac hypertrophy via suppressing NF-kappaB signalling, Cardiovasc. Res. 84 (2009) 119e126. [10] T. Yoshida, K. Abe, T. Ikeda, T. Matsushita, K. Wake, T. Sato, et al., Inhibitory effect of glycyrrhizin on lipopolysaccharide and d-galactosamine-induced mouse liver injury, Eur. J. Pharmacol. 576 (2007) 136e142. [11] Y.F. Ni, J.K. Kuai, Z.F. Lu, G.D. Yang, H.Y. Fu, J. Wang, et al., Glycyrrhizin treatment is associated with attenuation of lipopolysaccharide-induced acute lung injury by inhibiting cyclooxygenase-2 and inducible nitric oxide synthase expression, J. Surg. Res. 165 (2011) e29e35. [12] Y. Fu, E. Zhou, Z. Wei, D. Liang, W. Wang, T. Wang, et al., Glycyrrhizin inhibits the inflammatory response in mouse mammary epithelial cells and a mouse mastitis model, FEBS J. 281 (2014) 2543e2557. [13] Y.M. Kim, H.J. Kim, K.C. Chang, Glycyrrhizin reduces HMGB1 secretion in lipopolysaccharide-activated RAW 264.7 cells and endotoxemic mice by p38/ Nrf2-dependent induction of HO-1, Int. Immunopharmacol. 26 (2015) 112e118. [14] J.H. Song, J.W. Lee, B. Shim, C.Y. Lee, S. Choi, C. Kang, et al., Glycyrrhizin alleviates neuroinflammation and memory deficit induced by systemic lipopolysaccharide treatment in mice, Molecules 18 (2013) 15788e15803. [15] A. Zdarilova, A. Svobodova, V. Simanek, J. Ulrichova, Prunella vulgaris extract and rosmarinic acid suppress lipopolysaccharide-induced alteration in human
N. Zhang et al. / Microbial Pathogenesis 110 (2017) 135e139 gingival fibroblasts, Toxicol Vitro 23 (2009) 386e392. [16] C.A. Genco, T. Van Dyke, S. Amar, Animal models for Porphyromonas gingivalis-mediated periodontal disease, Trends Microbiol. 6 (1998) 444e449. [17] P.L. Wang, K. Sato, M. Oido, T. Fujii, Y. Kowashi, M. Shinohara, et al., Involvement of CD14 on human gingival fibroblasts in Porphyromonas gingivalis lipopolysaccharide-mediated interleukin-6 secretion, Archiv. oral Biol. 43 (1998) 687e694. [18] R. Assuma, T. Oates, D. Cochran, S. Amar, D.T. Graves, IL-1 and TNF antagonists inhibit the inflammatory response and bone loss in experimental periodontitis, J. Immunol. 160 (1998) 403e409. [19] T.E. Van Dyke, The management of inflammation in periodontal disease, J. Periodontol. 79 (2008) 1601e1608. [20] A. Chawla, J.J. Repa, R.M. Evans, D.J. Mangelsdorf, Nuclear receptors and lipid physiology: opening the X-files, Science 294 (2001) 1866e1870. [21] B.A. Janowski, M.J. Grogan, S.A. Jones, G.B. Wisely, S.A. Kliewer, E.J. Corey, et al., Structural requirements of ligands for the oxysterol liver X receptors LXRalpha and LXRbeta, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 266e271. [22] A.J. Fowler, M.Y. Sheu, M. Schmuth, J. Kao, J.W. Fluhr, L. Rhein, et al., Liver X receptor activators display anti-inflammatory activity in irritant and allergic contact dermatitis models: liver-X-receptor-specific inhibition of
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inflammation and primary cytokine production, J. investigative Dermatol. 120 (2003) 246e255. Y. Fu, X. Hu, Y. Cao, Z. Zhang, N. Zhang, Saikosaponin a inhibits lipopolysaccharide-oxidative stress and inflammation in Human umbilical vein endothelial cells via preventing TLR4 translocation into lipid rafts, Free Radic. Biol. Med. 89 (2015) 777e785. O. Cheng, R.P. Ostrowski, W. Liu, J.H. Zhang, Activation of liver X receptor reduces global ischemic brain injury by reduction of nuclear factor-kappaB, Neuroscience 166 (2010) 1101e1109. T. Crotti, M.D. Smith, R. Hirsch, S. Soukoulis, H. Weedon, M. Capone, et al., Receptor activator NF kappaB ligand (RANKL) and osteoprotegerin (OPG) protein expression in periodontitis, J. periodontal Res. 38 (2003) 380e387. P.L. Wang, Y. Azuma, M. Shinohara, K. Ohura, Toll-like receptor 4-mediated signal pathway induced by Porphyromonas gingivalis lipopolysaccharide in human gingival fibroblasts, Biochem. Biophys. Res. Commun. 273 (2000) 1161e1167. H.J. Park, S.K. Jeong, S.R. Kim, S.K. Bae, W.S. Kim, S.D. Jin, et al., Resveratrol inhibits Porphyromonas gingivalis lipopolysaccharide-induced endothelial adhesion molecule expression by suppressing NF-kappaB activation, Archiv. pharmacal Res. 32 (2009) 583e591.
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Inhibition of IL-6 and IL-8 production in LPS-stimulated human gingival fibroblasts by glycyrrhizin via activating LXRa Na Zhang a, b, Hui Lv b, Bo-Hong Shi b, Xin Hou b, Xin Xu a, * a b
Shandong Provincial Key Laboratory of Oral Tissue Regeneration, School of Stomatology, Shandong University, Shandong, China Department of Stomatology, The People's Hospital of Rizhao, Shandong, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 May 2017 Received in revised form 8 June 2017 Accepted 15 June 2017 Available online 23 June 2017
The aim of this study was to clarify the anti-inflammatory effects and its molecular mechanism of glycyrrhizin on LPS-stimulated human gingival fibroblasts (HGFs), which will be of benefit for periodontitis treatment. An MTT assay was performed to assess the effects of glycyrrhizin on cellular viability. The levels of IL-6 and IL-8 were measured by ELISA. The expression of iNOS, COX-2, NF-kB, and LXRa were detected by western blot analysis. The results showed that glycyrrhizin significantly inhibited LPSinduced IL-6 and IL-8 production, as well as COX-2 and iNOS expression. LPS-induced NF-kB activation in HGFs was also inhibited by treatment of glycyrrhizin. Furthermore, glycyrrhizin increased the expression of LXRa in a concentration-dependent manner. In addition, the inhibition of glycyrrhizin on IL-6 and IL-8 production was reversed by LXRa inhibitor GGPP. In conclusion, these results indicated that glycyrrhizin exhibited its anti-inflammatory effects in HGFs by activating LXRa. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Glycyrrhizin Human gingival fibroblasts IL-8 NF-kB LXRa
1. Introduction Periodontitis is a common and often undiagnosed oral disease that characterized by inflammatory destruction of periodontal tissues [1]. Periodontal destruction is often caused by bacteria, which leads to the inflammation and bone resorption [2]. Porphyromonas gingivalis is the major pathogen that leads to periodontitis [3,4]. Porphyromonas gingivalis LPS could induce NF-kB activation and inflammatory mediators release in human gingival fibroblasts [5]. These inflammatory mediators, such as IL-6, IL-8, NO, and PGE2, have the ability to induce periodontal tissue injury [6]. Previous studies showed that inhibition of inflammatory response could attenuate the pathological process of periodontitis [7]. LXRa is a nuclear receptor that has previously been shown to regulate inflammatory response [8]. It has been reported that activating LXRa could inhibit LPS-induced NF-kB activation [9]. Therefore, LXRa may be used as a target for the treatment of periodontitis. Glycyrrhizin, an active component of liquorice roots, has been reported to have anti-inflammatory effect. Previous study showed that glycyrrhizin inhibited LPS and D-galactosamine-induced liver injury in mice [10]. Glycyrrhizin also suppressed LPS-induced acute
* Corresponding author. E-mail address: [email protected] (X. Xu). http://dx.doi.org/10.1016/j.micpath.2017.06.021 0882-4010/© 2017 Elsevier Ltd. All rights reserved.
lung injury in mice by inhibiting inflammatory mediators production [11]. Glycyrrhizin was found to inhibit LPS-induced inflammatory cytokines production in mammary epithelial cells [12]. Moreover, glycyrrhizin has been known to inhibit HMGB1 secretion in LPS-stimulated RAW264.7 cells [13]. LPS-induced neuroinflammation and memory deficit were also inhibited by treatment of glycyrrhizin [14]. However, the anti-inflammatory effects of glycyrrhizin on LPS-induced inflammatory response in HGFs have not been reported. The aim of this study was to clarify the antiinflammatory effects and mechanism of glycyrrhizin in LPSstimulated HGFs. 2. Materials and methods 2.1. Chemicals and reagents Glycyrrhizin (purity>98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). GGPP was purchased from Sigma-Aldrich (CA, USA). LPS from Porphyromonas gingivalis was obtained from InvivoGen (San Diego, CA, USA). NF-kB p65, NF-kB p-p65, IkBa, p-IkBa, iNOS, COX-2, and b-actin were purchased from Cell Signaling Technology Inc (Beverly, MA). LXRa antibody was purchased from Santa Cruz Biotechnology. ELISA kits for IL-6 and IL-8 were purchased from R&D. All other reagents were of analytical grade.
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2.2. Cell culture HGFs were isolated from explants of human normal gingival tissues as described previously [15]. This study was approved by the local ethic committee. The cells were suspended in DMEM containing 10% fetal bovine serum (FBS). Cells between passages 3 to 8 were used in this study. For LXRa inhibitory experiment, the cells were pretreated with or without 20 mM geranylgeranyl pyrophosphate (GGPP) for 2 h, then the cells were pretreated with glycyrrhizin 12 h before LPS treatment. 2.3. Cell viability Cell viability was detected by MTT assay. In brief, HGFs were seeded at a 96-well plate at the density of 1 104 cells/well. Then, the cells were treated with LPS and glycyrrhizin for 24 h. MTT was added to each well. 4 h later, 150 mL of DMSO was added to each well. Optical density was measured at 450 nm using a Bio-Rad Microplate Reader (Model 680, Bio-Rad, USA).
Fig. 1. Effects of glycyrrhizin on the cell viability of HGFs. Cells were cultured with different concentrations of glycyrrhizin (0e180 mg/ml) in the absence or presence of 1 mg/ml LPS for 24 h. The cell viability was determined by MTT assay. The values presented are the means ± SD of three independent experiments.
2.4. Cytokines assay The culture medium was collected and the production of cytokines IL-6 and IL-8 in the culture medium were measured using ELISA kits (R&D) according to the manufacturer's instructions. 2.5. Western blot analysis HGFs were lysed using RIPA lysis buffer and the protein concentration was detected by BCA method. The protein (30 mg) were separated by 12% SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad, PA, USA). The membranes were incubated with antibodies against COX-2, iNOS, NF-kB p65, NF-kB p-p65, IkBa, p-IkBa, LXRa, and b-actin overnight at 4 C. Then, the membranes were probed with HRP-conjugated secondary antibody for 1 h. The specific binding was detected using chemiluminescence detection system (Amersham, Berkshire, UK). 2.6. Statistical analysis Results were presented as means ± SD. Differences between multiple treatments were analyzed with one-way analysis followed by the Newman-Keuls post hoc test. Statistically significance were considered at P < 0.05. 3. Results 3.1. Effects of glycyrrhizin on HGFs cell viability Effects of glycyrrhizin on HGFs cell viability was assessed by the MTT assay. As shown in Fig. 1, glycyrrhizin at the doses of 40, 80, 160 mg/ml did not affect the cell viabilities of HGFs. Glycyrrhizin at the dose of 180 mg/ml decreased the cell viabilities of HGFs. Thus, in the subsequent studies, glycyrrhizin at the doses of 40, 80, 160 mg/ ml were used. 3.2. Glycyrrhizin inhibits LPS-induced IL-6 and IL-8 production in HGFs To evaluate the anti-inflammatory effects of glycyrrhizin on LPSstimulated HGFs, the production of IL-6 and IL-8 were detected. As shown in Fig. 2, LPS treatment increased the production of IL-6 and IL-8 compared with the untreated group. However, pre-treatment of the cells with glycyrrhizin decreased the production of IL-6 and IL-8 compared with the LPS group.
Fig. 2. Glycyrrhizin inhibits LPS-induced IL-8 and IL-6 production in HGFs. The data presented are the means ± SD of three independent experiments. #p < 0.05 vs. control group; *p < 0.05, **p < 0.01 vs. LPS group.
3.3. Glycyrrhizin inhibits LPS-induced COX-2 and iNOS expression in HGFs To evaluate the anti-inflammatory effects of glycyrrhizin on LPSstimulated HGFs, the production of IL-6 and IL-8 were detected. As shown in Fig. 3, LPS treatment increased the production of IL-6 and IL-8 compared with the untreated group. However, pre-treatment of the cells with glycyrrhizin decreased the production of IL-6 and IL-8 compared with the LPS group.
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Fig. 3. Effects of glycyrrhizin on iNOS and COX-2 expression in LPS-stimulated HGFs. The data presented are the means ± SD of three independent experiments. #p < 0.05 vs. control group; *p < 0.05, **p < 0.01 vs. LPS group.
3.4. Glycyrrhizin inhibits LPS-induced NF-kB activation in HGFs NF-kB is essential for the expression of inflammatory mediators. Thus, we investigated whether glycyrrhizin could inhibit LPSinduced NF-kB activation. As shown in Fig. 4, LPS treatment induced IkB-a phosphorylation and degradation and NF-kB p65 phosphorylation. However, treatment of glycyrrhizin significantly inhibited LPS-induced IkB-a phosphorylation and degradation and NF-kB p65 phosphorylation. 3.5. Glycyrrhizin exhibits anti-inflammatory effects in HGFs by activating LXRa To further investigate the anti-inflammatory mechanism of glycyrrhizin, the effects of glycyrrhizin on LXRa expression were
detected in this study. As shown in Fig. 5A, glycyrrhizin concentration-dependently up-regulated the expression of LXRa. Furthermore, our results showed that the inhibition of glycyrrhizin on IL-6 and IL-8 production can be reversed by LXRa inhibitor GGPP (Fig. 5B). 4. Discussion In the present study, we designed to detect whether glycyrrhizin could inhibit LPS-induced inflammatory response in HGFs, which has been associated with the development of periodontal diseases. We found that glycyrrhizin inhibited LPS-induced inflammatory mediators production by activating LXRa. Porphyromonas gingivalis is a major pathogen of periodontal disease that leads to inflammatory response in the gingival [16].
Fig. 4. Effects of glycyrrhizin on LPS-induced NF-kB activation and IkBa degradation. The values presented are the means ± SD of three independent experiments. #p < 0.05 vs. control group; *p < 0.05, **p < 0.01 vs. LPS group.
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effects in LPS-stimulated HGFs. LXRa is a member of the nuclear hormone receptor superfamily that has been reported to regulate the metabolism of lipids [20,21]. A large body of studies showed that LXR agonists possessed antiinflammatory properties [22,23]. Activation of LXRa could attenuate the activation of transcription factor NF-kB [24]. Studies showed that activation of NF-kB contributed to the pathologic process of periodontitis [25]. Stimulating of HGFs with LPS could induce the activation of NF-kB [26]. Previous studies showed that inhibition of NF-kB activation could attenuate the pathologic process of periodontitis [27]. In the present study, our results showed that glycyrrhizin significantly inhibited LPS-induced NF-kB activation. In addition, we found that glycyrrhizin concentrationdependently up-regulated the expression of LXRa. The antiinflammatory effects of glycyrrhizin were blocked by LXRa inhibitor GGPP. These results suggested that glycyrrhizin inhibited LPSinduced inflammatory response in HGFs by activating LXRa. In conclusion, our results found that glycyrrhizin significantly suppressed LPS-induced IL-6 and IL-8 production in HGFs. The antiinflammatory mechanism was through activating LXRa, which subsequently inhibited LPS-induced NF-kB activation. These results suggested that glycyrrhizin may provide a beneficial effect for treating inflammation and periodontal diseases. Conflict of interest All authors declare that they have no conflict of interest. References
Fig. 5. (A) Effects of glycyrrhizin on LXRa expression. (B) Block LXRa reversed the antiinflammatory effects of glycyrrhizin. The values presented are the means ± SD of three independent experiments. #P < 0.05 vs. control group; *P < 0.05, **P < 0.01 vs. LPS group.
Stimulation of gingival fibroblasts by Porphyromonas gingivalis LPS induced the release of inflammatory mediators [17]. These inflammatory mediators played critical roles in the development of periodontitis [1]. Previous studies showed that inhibition of these inflammatory mediators might attenuate the pathologic process of periodontitis [18,19]. To explore the anti-inflammatory effects of glycyrrhizin, the effects of glycyrrhizin on LPS-induced inflammatory mediators production were detected. Our results demonstrated that glycyrrhizin significantly inhibited LPS-induced IL-6 and IL-8 production, as well as COX-2 and iNOS expression. The results suggested that glycyrrhizin exhibited anti-inflammatory
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