- Email: [email protected]
AKT signaling pathway
Accepted Manuscript Plantamajoside attenuates inflammatory response in LPS-stimulated human gingival fibroblasts by inhibiting PI3K/AKT signaling pathway Fei Liu, Xin Huang, Jing-jun He, Ci Song, Ling Peng, Ting Chen, Bu-ling Wu PII:
S0882-4010(18)31156-2
DOI:
https://doi.org/10.1016/j.micpath.2018.11.034
Reference:
YMPAT 3271
To appear in:
Microbial Pathogenesis
Received Date: 26 June 2018 Revised Date:
14 November 2018
Accepted Date: 22 November 2018
Please cite this article as: Liu F, Huang X, He J-j, Song C, Peng L, Chen T, Wu B-l, Plantamajoside attenuates inflammatory response in LPS-stimulated human gingival fibroblasts by inhibiting PI3K/AKT signaling pathway, Microbial Pathogenesis (2018), doi: https://doi.org/10.1016/j.micpath.2018.11.034. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Plantamajoside attenuates inflammatory response in LPS-stimulated human gingival fibroblasts by inhibiting PI3K/AKT signaling pathway Fei Liu1,2,3#, Xin Huang4#, Jing-jun He3, Ci Song1,2, Ling Peng1,2, Ting Chen1,2*, Bu-ling Wu1,2* 1. Department of Stomatology, Nanfang Hospital, Guangzhou, 510515, China. 2. College of Stomatology, Southern Medical University, Guangzhou, 510515, China. 3. International medical center, Guangdong Second Provincial General Hospital, Guangzhou, 510010, China. 4. Department of Stomatology, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, 510515, China. # Fei Liu and Xin Huang are co-first authors. * Correspondence to: Ting Chen, E-mail: [email protected]; Bu-ling Wu, E-mail: [email protected].
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Abstract Periodontitis is an important inflammatory disease that often causes by periodontopathic bacteria. The present study, we tested the anti-inflammatory effects of plantamajoside on LPS-stimulated human gingival fibroblasts. Human gingival fibroblasts (HGFs) were stimulated with LPS from Porphyromonas gingivalis. Plantamajoside was administrated 1 h before LPS treatment. The results demonstrated that plantamajoside decreased the production of PGE2, NO, IL-6, and IL-8 in LPS-stimulated HGFs. LPS-induced NF-κB p65 and IκB phosphorylation were also suppressed by plantamajoside. Furthermore, plantamajoside inhibited LPS-induced PI3K and AKT phosphorylation. In conclusion, these results suggested that the mechanism of plantamajoside was through inhibiting PI3K/AKT signaling pathway, which lead to the inhibition of NF-κB activation and inflammatory response. Keywords: plantamajoside; LPS; human gingival fibroblasts; IL-8; PI3K
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1. Introduction Periodontitis is an inflammatory disease of periradicular tissues that often affects millions of people [1]. It is characterized by inflammation of the supporting tissues of the teeth [2]. Although the pathogenesis of periodontitis is complex, it is widely accepted that bacterial infection is the major cause [3]. Gingival fibroblasts respond to oral LPS, the outer membrane component of Gram-negative bacteria, by releasing a large body of inflammatory mediators [4]. Previous studies demonstrated that there was a relationship between systemic inflammation and periodontitis [5, 6]. Elevated inflammatory mediators, such as PGE2, NO, and IL-6 were observed in patients of periodontitis [7]. These inflammatory mediators could induce the destruction of periodontal tissues [8]. Therefore, to attenuate the inflammatory response is helpful for the treatment of periodontitis. Plantamajoside is a phenylpropanoid glycoside isolated from Plantago asiatica [9]. Recently, it has been reported to have anti-inflammatory effects [10]. Plantamajoside was found to inhibit LPS-induced epithelial-mesenchymal transition via inhibiting NF-κB activation [11]. Plantamajoside also inhibited LPS-induced inflammation in human airway epithelial cells [12]. Furthermore, it has been reported plantamajoside had protective effects against LPS-induced acute lung injury [12]. In addition, plantamajoside was found to regulate human umbilical vein
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endothelial cell dysfunction through inhibiting MAPK and NF-κB signaling pathways [13]. Elevated inflammatory mediators were observed in patients of periodontitis and studies showed that inhibition of the inflammatory response could attenuate the pathogenesis of periodontitis [14]. Human gingival fibroblasts are the major cell model that used to investigate the inflammation of periodontitis. In this study, we investigated the anti-inflammatory effects of plantamajoside on periodontitis using the in vitro model. We investigated the anti-inflammatory effects and mechanism of plantamajoside on LPS-stimulated HGFs in vitro. 2. Materials and methods 2.1. Chemicals and reagents
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LPS from Porphyromonas gingivalis was obtained from InvivoGen (San Diego, CA, USA). Plantamajoside was purchased from National Institutes for Food and Drug Control (Beijing, China). MTT and DMSO were purchased from Sigma (St. Louis, MO, USA). ELISA kits for TNF-α, IL-1β, and PGE2 were purchased from Biolegend (CA, USA). Antibodies for p-PI3K, PI3K, p-AKT, AKT, NF-κB p65, IκBα, NF-κB p-p65, p-IκBα, and β-actin were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA).
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2.2. Cell culture HGFs were cultured from explants of human normal gingival tissues as described previously and cultured in DMEM containing 10% FBS containing and 1% penicillin/streptomycin (100 U/ml penicillin and 100 µg/ml streptomycin) at 37 °C with 5% CO2. The explants were obtained from patients undergoing surgery for the removal of third impacted molars. Informed consent was obtained from all donors. The experiment was in accordance with the Declaration of Helsinki and Tokyo. HGFs were used between the 5th and 10th passages.
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2.3. Cell viability HGFs were pre-treated with different concentrations of plantamajoside and stimulated with LPS for 18 h. Then, MTT (5 mg/ml) was added to each well for 4 h. Subsequently, 150 µl of DMSA was added to each well. Finally, the optical density (D) value was detected at 570 nm using a Bio-Rad Microplate Reader (Model 680, Bio-Rad, USA). 2.4. Inflammatory mediators assay
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24 h after LPS treatment, the supernatants were collected. The levels of IL-6, IL-8, and PGE2 were tested using commercially available ELISA kits (Biolegend, USA) according to the manufacturer’s instructions. The level of nitrite in the supernatant was tested using the Griess reaction (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. 2.5. Western blot analysis The cells were collected and lysed with RIPA buffer and protein concentration was measured by BCA method. The proteins (30µg) were separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat milk and washed with TBST for three times. Then, the membranes were incubated overnight with primary antibodies (1:1000 dilutions in TBST): PI3K, AKT, NF-κB p65, and IκBα. After washing three times with TBST, the membranes were probed with 1 : 2000 (v/v) dilution of HRP-conjugated secondary antibody. Finally, the bands were visualized using enhanced chemiluminescence reagents (ECL).
ACCEPTED MANUSCRIPT The bands were analyzed using the ImageJ 1.39v software (Wayne Rasband National Institutes of Health).
3.1. Effects of plantamajoside on cell viability
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2.6. Statistical analysis The values were presented as means ± SEM of three separate experiments. Different between goups were analyzed using one-way ANOVA followed by Dunnett’s test using the SPSS software. All statistical tests with P < 0.05 were considered significantly different. 3. Results
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The cytotoxicity of plantamajoside on HGFs was measured by MTT assay. The results showed plantamajoside at the concentration of 0-40µg/ml did not affect the cell viability of HGFs. Thus, we chose the concentration of 10, 20, and 40µg/ml in the following studies (Fig. 1). 3.2. Plantamajoside inhibits PGE2 and NO production following LPS treatment
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PGE2 and NO are important inflammatory mediators that involved in the pathogenesis of periodontitis. The present study, we investigated the effects of plantamajoside on PGE2 and NO production following LPS treatment. The exposure of HGFs to LPS for 24 h significantly increased the production of PGE2 and NO. However, treatment of plantamajoside inhibited LPS-induced PGE2 and NO production in a concentration-dependent manner (Fig. 2). 3.3. Plantamajoside inhibits IL-6 and IL-8 production following LPS treatment
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IL-6 and IL-8 are important cytokines that involved in the pathogenesis of periodontitis. The present study, we investigated the effects of plantamajoside on IL-6 and IL-8 production following LPS treatment. The exposure of HGFs to LPS for 24 h significantly increased the production of IL-6 and IL-8. However, treatment of plantamajoside inhibited LPS-induced IL-6 and IL-8 production in a concentration-dependent manner (Fig. 3). 3.4. Plantamajoside inhibits NF-κB activation following LPS treatment
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To investigate the anti-inflammatory mechanism of plantamajoside, NF-κB activation was measured by western blot analysis. The exposure of HGFs to LPS significantly increased the phosphorylation levels of NF-κB p65 and IκBα. However, treatment of plantamajoside inhibited LPS-induced NF-κB activation in a concentration-dependent manner (Fig. 4). 3.5. Plantamajoside inhibits PI3K and AKT phosphorylation following LPS treatment To identify the upstream molecules by which plantamajoside mediated the inhibition of NF-κB activation, PI3K and AKT phosphorylation were detected in this study. The exposure of HGFs to LPS significantly increased the phosphorylation levels of PI3K and AKT. However, treatment of plantamajoside inhibited LPS-induced PI3K and AKT phosphorylation in a concentration-dependent manner (Fig. 5). 4. Discussion In this study, our results showed that plantamajoside, a phenylpropanoid glycoside isolated from Plantago asiatica, had anti-inflammatory effects against LPS-stimulated HGFs. The mechanism was through inhibiting LPS-induced PI3K/AKT/NF-κB signaling pwathway. Plantamajoside may have protective effects against periodontitis.
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Gingival fibroblasts have been known as the major cells for periodontitis [15]. In the development of periodontitis, LPS from Gram-negative bacteria could induce the production of inflammatory mediators [16]. The excess expression of PGE2 and NO following gingival fibroblasts activation contributed to the development of periodontitis [17]. Furthermore, the release of cytokine IL-8 could induce the recruitment of neutrophils, which subsequently release the inflammatory mediators and amplify the inflammatory response [18]. Elevated PGE2, NO, IL-6, and IL-8 were observed in patients of periodontitis [19]. PGE2 was a key inflammatory mediator involved in periodontitis. It is a strong stimulator of bone resorption and formation. It could stimulate collagen synthesis and regulate bone density [20]. NO could stimulate cyclooxygenase and metalloproteinases which may result in periodontal tissue damage. IL-6 was a powerful stimulator of osteoclast differentiation and bone resorption [21]. Therefore, inhibition of these inflammatory mediators could attenuate the damage of the supporting tissues of the teeth and protect periodontitis. In this study, our results showed that plantamajoside significantly reduced the production of these inflammatory mediators. These results indicated that plantamajoside had anti-inflammatory activity against periodontitis and might have protective effects against periodontitis. NF-κB is associated with the expression of inflammatory mediators. Activation of NF-κB could induce the production of inflammatory mediators [22]. Emerging evidence demonstrated that LPS from Porphyromonas gingivalis could induce the activation of NF-κB [23]. And NF-κB has been identified as an important target for the treatment of periodontitis [24]. Furthermore, inhibition of NF-κB activation could attenuate the pathologic process of periodontitis [25]. The present study we found plantamajoside significantly attenuated LPS-induced NF-κB activation. To identify the upstream signaling by which plantamajoside mediated the inhibition of NF-κB activation, PI3K/AKT signaling pathway was measured in this study. Our results showed that plantamajoside significantly attenuated LPS-induced PI3K and AKT phosphorylation. In summary, the results of this study showed that plantamajoside inhibited PI3K/AKT signaling pathway, which subsequently lead to the inhibition of NF-κB activation and inflammatory mediators production. Plantamajoside may be used as a potential candidate for the treatment of periodontitis. Conflict of interest statement
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All authors declare that they have no conflict of interest. References
[1] Chapple IL, Genco R. Diabetes and periodontal diseases: consensus report of the Joint EFP/AAP Workshop on Periodontitis and Systemic Diseases. Journal of periodontology. 2013;84:S106-12. [2] Di Benedetto A, Gigante I, Colucci S, Grano M. Periodontal disease: linking the primary inflammation to bone loss. Clinical & developmental immunology. 2013;2013:503754. [3] Li X, Kolltveit KM, Tronstad L, Olsen I. Systemic diseases caused by oral infection. Clinical microbiology reviews. 2000;13:547-58. [4] Wang PL, Ohura K. Porphyromonas gingivalis lipopolysaccharide signaling in gingival fibroblasts-CD14 and Toll-like receptors. Critical reviews in oral biology and medicine : an official publication of the American Association of Oral Biologists. 2002;13:132-42. [5] Loos BG. Systemic markers of inflammation in periodontitis. Journal of periodontology.
ACCEPTED MANUSCRIPT 2005;76:2106-15. [6] Shi D, Meng H, Xu L, Zhang L, Chen Z, Feng X, et al. Systemic inflammation markers in patients with aggressive periodontitis: a pilot study. Journal of periodontology. 2008;79:2340-6. [7] D'Aiuto F, Nibali L, Parkar M, Patel K, Suvan J, Donos N. Oxidative stress, systemic inflammation, and severe periodontitis. Journal of dental research. 2010;89:1241-6. [8] Graves DT, Cochran D. The contribution of interleukin-1 and tumor necrosis factor to periodontal tissue destruction. Journal of periodontology. 2003;74:391-401.
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[9] Li Y, Gan L, Li GQ, Deng L, Zhang X, Deng Y. Pharmacokinetics of plantamajoside and acteoside from Plantago asiatica in rats by liquid chromatography-mass spectrometry. Journal of pharmaceutical and biomedical analysis. 2014;89:251-6.
[10] Ma C, Ma W. Plantamajoside Inhibits Lipopolysaccharide-Induced MUC5AC Expression and Inflammation through Suppressing the PI3K/Akt and NF-kappaB Signaling Pathways in Human
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[11] Li X, Chen D, Li M, Gao X, Shi G, Zhao H. Plantamajoside inhibits lipopolysaccharide-induced epithelial-mesenchymal transition through suppressing the NF-kappaB/IL-6 signaling in esophageal squamous cell carcinoma cells. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie.
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[12] Wu H, Zhao G, Jiang K, Chen X, Zhu Z, Qiu C, et al. Plantamajoside ameliorates lipopolysaccharide-induced acute lung injury via suppressing NF-kappaB and MAPK activation. International immunopharmacology. 2016;35:315-22.
[13] Son WR, Nam MH, Hong CO, Kim Y, Lee KW. Plantamajoside from Plantago asiatica modulates human umbilical vein endothelial cell dysfunction by glyceraldehyde-induced AGEs via MAPK/NF-kappaB. BMC complementary and alternative medicine. 2017;17:66.
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[14] D'Aiuto F, Parkar M, Andreou G, Suvan J, Brett PM, Ready D, et al. Periodontitis and systemic inflammation: Control of the local infection is associated with a reduction in serum inflammatory markers. Journal of dental research. 2004;83:156-60. [15] Genco RJ. Host Responses in Periodontal Diseases: Current Concepts. Journal of periodontology. 1992;63 Suppl 4S:338-55.
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[16] Sun Y, Shu R, Li CL, Zhang MZ. Gram-negative periodontal bacteria induce the activation of Toll-like receptors 2 and 4, and cytokine production in human periodontal ligament cells. Journal of periodontology. 2010;81:1488-96.
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[17] Kornman KS, di Giovine FS. Genetic variations in cytokine expression: a risk factor for severity of adult periodontitis. Annals of periodontology. 1998;3:327-38. [18] Harada A, Sekido N, Akahoshi T, Wada T, Mukaida N, Matsushima K. Essential involvement of interleukin-8 (IL-8) in acute inflammation. Journal of leukocyte biology. 1994;56:559-64. [19] Yucel-Lindberg T, Bage T. Inflammatory mediators in the pathogenesis of periodontitis. Expert Rev Mol Med. 2013;15.
[20] Garrison SW, Holt SC, Nichols FC. Lipopolysaccharide-stimulated PGE2 release from human monocytes. Comparison of lipopolysaccharides prepared from suspected periodontal pathogens. J Periodontol. 1988;59:684-7. [21] Reinhardt RA, Masada MP, Kaldahl WB, DuBois LM, Kornman KS, Choi JI, et al. Gingival fluid IL-1 and IL-6 levels in refractory periodontitis. Journal of clinical periodontology. 1993;20:225-31. [22] Lawrence T, Gilroy DW, Colville-Nash PR, Willoughby DA. Possible new role for NF-kappaB in the resolution of inflammation. Nature medicine. 2001;7:1291-7.
ACCEPTED MANUSCRIPT [23] Herath TD, Darveau RP, Seneviratne CJ, Wang CY, Wang Y, Jin L. Tetra- and 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. 2013;8:e58496. [24] Jimi E, Aoki K, Saito H, D'Acquisto F, May MJ, Nakamura I, et al. Selective inhibition of NF-kappa B blocks osteoclastogenesis and prevents inflammatory bone destruction in vivo. Nature medicine. 2004;10:617-24.
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[25] Nichols TC, Fischer TH, Deliargyris EN, Baldwin AS, Jr. Role of nuclear factor-kappa B (NF-kappa B) in inflammation, periodontitis, and atherogenesis. Annals of periodontology. 2001;6:20-9.
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Figure Legends Figure 1. Effects of plantamajoside on the cell viability of HGFs. The cell viability was determined by MTT assay. The values presented are the means ± SEM of three independent experiments.
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Figure 2. Plantamajoside inhibits LPS-induced PGE2 and NO production in HGFs. The data presented are the means ± SEM of three independent experiments. #p < 0.05 vs. control group; *p < 0.05, **p < 0.01 vs. LPS group. Figure 3. Plantamajoside inhibits LPS-induced IL-6 and IL-8 production in HGFs. The data presented are the means ± SEM of three independent experiments. #p < 0.05 vs. control group; *p < 0.05, **p < 0.01 vs. LPS group.
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Figure 4. Effects of plantamajoside on LPS-induced NF-κB activation and IκBα degradation. The values presented are the means ± SEM of three independent experiments. #p < 0.05 vs. control group; *p < 0.05, **p < 0.01 vs. LPS group.
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Figure 5. Effects of plantamajoside on LPS-induced PI3K/AKT signaling pathway. The values presented are the means ± SEM of three independent experiments. #p < 0.05 vs. control group; *p < 0.05, **p < 0.01 vs. LPS group.
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Plantamajoside decreased the production of PGE2, NO, IL-6, and IL-8 in LPS-stimulated HGFs. LPS-induced NF-κB p65 and IκB phosphorylation were also suppressed by plantamajoside. Plantamajoside inhibited LPS-induced PI3K and AKT phosphorylation.
S0882-4010(18)31156-2
DOI:
https://doi.org/10.1016/j.micpath.2018.11.034
Reference:
YMPAT 3271
To appear in:
Microbial Pathogenesis
Received Date: 26 June 2018 Revised Date:
14 November 2018
Accepted Date: 22 November 2018
Please cite this article as: Liu F, Huang X, He J-j, Song C, Peng L, Chen T, Wu B-l, Plantamajoside attenuates inflammatory response in LPS-stimulated human gingival fibroblasts by inhibiting PI3K/AKT signaling pathway, Microbial Pathogenesis (2018), doi: https://doi.org/10.1016/j.micpath.2018.11.034. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Plantamajoside attenuates inflammatory response in LPS-stimulated human gingival fibroblasts by inhibiting PI3K/AKT signaling pathway Fei Liu1,2,3#, Xin Huang4#, Jing-jun He3, Ci Song1,2, Ling Peng1,2, Ting Chen1,2*, Bu-ling Wu1,2* 1. Department of Stomatology, Nanfang Hospital, Guangzhou, 510515, China. 2. College of Stomatology, Southern Medical University, Guangzhou, 510515, China. 3. International medical center, Guangdong Second Provincial General Hospital, Guangzhou, 510010, China. 4. Department of Stomatology, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, 510515, China. # Fei Liu and Xin Huang are co-first authors. * Correspondence to: Ting Chen, E-mail: [email protected]; Bu-ling Wu, E-mail: [email protected].
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Abstract Periodontitis is an important inflammatory disease that often causes by periodontopathic bacteria. The present study, we tested the anti-inflammatory effects of plantamajoside on LPS-stimulated human gingival fibroblasts. Human gingival fibroblasts (HGFs) were stimulated with LPS from Porphyromonas gingivalis. Plantamajoside was administrated 1 h before LPS treatment. The results demonstrated that plantamajoside decreased the production of PGE2, NO, IL-6, and IL-8 in LPS-stimulated HGFs. LPS-induced NF-κB p65 and IκB phosphorylation were also suppressed by plantamajoside. Furthermore, plantamajoside inhibited LPS-induced PI3K and AKT phosphorylation. In conclusion, these results suggested that the mechanism of plantamajoside was through inhibiting PI3K/AKT signaling pathway, which lead to the inhibition of NF-κB activation and inflammatory response. Keywords: plantamajoside; LPS; human gingival fibroblasts; IL-8; PI3K
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1. Introduction Periodontitis is an inflammatory disease of periradicular tissues that often affects millions of people [1]. It is characterized by inflammation of the supporting tissues of the teeth [2]. Although the pathogenesis of periodontitis is complex, it is widely accepted that bacterial infection is the major cause [3]. Gingival fibroblasts respond to oral LPS, the outer membrane component of Gram-negative bacteria, by releasing a large body of inflammatory mediators [4]. Previous studies demonstrated that there was a relationship between systemic inflammation and periodontitis [5, 6]. Elevated inflammatory mediators, such as PGE2, NO, and IL-6 were observed in patients of periodontitis [7]. These inflammatory mediators could induce the destruction of periodontal tissues [8]. Therefore, to attenuate the inflammatory response is helpful for the treatment of periodontitis. Plantamajoside is a phenylpropanoid glycoside isolated from Plantago asiatica [9]. Recently, it has been reported to have anti-inflammatory effects [10]. Plantamajoside was found to inhibit LPS-induced epithelial-mesenchymal transition via inhibiting NF-κB activation [11]. Plantamajoside also inhibited LPS-induced inflammation in human airway epithelial cells [12]. Furthermore, it has been reported plantamajoside had protective effects against LPS-induced acute lung injury [12]. In addition, plantamajoside was found to regulate human umbilical vein
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endothelial cell dysfunction through inhibiting MAPK and NF-κB signaling pathways [13]. Elevated inflammatory mediators were observed in patients of periodontitis and studies showed that inhibition of the inflammatory response could attenuate the pathogenesis of periodontitis [14]. Human gingival fibroblasts are the major cell model that used to investigate the inflammation of periodontitis. In this study, we investigated the anti-inflammatory effects of plantamajoside on periodontitis using the in vitro model. We investigated the anti-inflammatory effects and mechanism of plantamajoside on LPS-stimulated HGFs in vitro. 2. Materials and methods 2.1. Chemicals and reagents
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LPS from Porphyromonas gingivalis was obtained from InvivoGen (San Diego, CA, USA). Plantamajoside was purchased from National Institutes for Food and Drug Control (Beijing, China). MTT and DMSO were purchased from Sigma (St. Louis, MO, USA). ELISA kits for TNF-α, IL-1β, and PGE2 were purchased from Biolegend (CA, USA). Antibodies for p-PI3K, PI3K, p-AKT, AKT, NF-κB p65, IκBα, NF-κB p-p65, p-IκBα, and β-actin were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA).
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2.2. Cell culture HGFs were cultured from explants of human normal gingival tissues as described previously and cultured in DMEM containing 10% FBS containing and 1% penicillin/streptomycin (100 U/ml penicillin and 100 µg/ml streptomycin) at 37 °C with 5% CO2. The explants were obtained from patients undergoing surgery for the removal of third impacted molars. Informed consent was obtained from all donors. The experiment was in accordance with the Declaration of Helsinki and Tokyo. HGFs were used between the 5th and 10th passages.
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2.3. Cell viability HGFs were pre-treated with different concentrations of plantamajoside and stimulated with LPS for 18 h. Then, MTT (5 mg/ml) was added to each well for 4 h. Subsequently, 150 µl of DMSA was added to each well. Finally, the optical density (D) value was detected at 570 nm using a Bio-Rad Microplate Reader (Model 680, Bio-Rad, USA). 2.4. Inflammatory mediators assay
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24 h after LPS treatment, the supernatants were collected. The levels of IL-6, IL-8, and PGE2 were tested using commercially available ELISA kits (Biolegend, USA) according to the manufacturer’s instructions. The level of nitrite in the supernatant was tested using the Griess reaction (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. 2.5. Western blot analysis The cells were collected and lysed with RIPA buffer and protein concentration was measured by BCA method. The proteins (30µg) were separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat milk and washed with TBST for three times. Then, the membranes were incubated overnight with primary antibodies (1:1000 dilutions in TBST): PI3K, AKT, NF-κB p65, and IκBα. After washing three times with TBST, the membranes were probed with 1 : 2000 (v/v) dilution of HRP-conjugated secondary antibody. Finally, the bands were visualized using enhanced chemiluminescence reagents (ECL).
ACCEPTED MANUSCRIPT The bands were analyzed using the ImageJ 1.39v software (Wayne Rasband National Institutes of Health).
3.1. Effects of plantamajoside on cell viability
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2.6. Statistical analysis The values were presented as means ± SEM of three separate experiments. Different between goups were analyzed using one-way ANOVA followed by Dunnett’s test using the SPSS software. All statistical tests with P < 0.05 were considered significantly different. 3. Results
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The cytotoxicity of plantamajoside on HGFs was measured by MTT assay. The results showed plantamajoside at the concentration of 0-40µg/ml did not affect the cell viability of HGFs. Thus, we chose the concentration of 10, 20, and 40µg/ml in the following studies (Fig. 1). 3.2. Plantamajoside inhibits PGE2 and NO production following LPS treatment
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PGE2 and NO are important inflammatory mediators that involved in the pathogenesis of periodontitis. The present study, we investigated the effects of plantamajoside on PGE2 and NO production following LPS treatment. The exposure of HGFs to LPS for 24 h significantly increased the production of PGE2 and NO. However, treatment of plantamajoside inhibited LPS-induced PGE2 and NO production in a concentration-dependent manner (Fig. 2). 3.3. Plantamajoside inhibits IL-6 and IL-8 production following LPS treatment
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IL-6 and IL-8 are important cytokines that involved in the pathogenesis of periodontitis. The present study, we investigated the effects of plantamajoside on IL-6 and IL-8 production following LPS treatment. The exposure of HGFs to LPS for 24 h significantly increased the production of IL-6 and IL-8. However, treatment of plantamajoside inhibited LPS-induced IL-6 and IL-8 production in a concentration-dependent manner (Fig. 3). 3.4. Plantamajoside inhibits NF-κB activation following LPS treatment
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To investigate the anti-inflammatory mechanism of plantamajoside, NF-κB activation was measured by western blot analysis. The exposure of HGFs to LPS significantly increased the phosphorylation levels of NF-κB p65 and IκBα. However, treatment of plantamajoside inhibited LPS-induced NF-κB activation in a concentration-dependent manner (Fig. 4). 3.5. Plantamajoside inhibits PI3K and AKT phosphorylation following LPS treatment To identify the upstream molecules by which plantamajoside mediated the inhibition of NF-κB activation, PI3K and AKT phosphorylation were detected in this study. The exposure of HGFs to LPS significantly increased the phosphorylation levels of PI3K and AKT. However, treatment of plantamajoside inhibited LPS-induced PI3K and AKT phosphorylation in a concentration-dependent manner (Fig. 5). 4. Discussion In this study, our results showed that plantamajoside, a phenylpropanoid glycoside isolated from Plantago asiatica, had anti-inflammatory effects against LPS-stimulated HGFs. The mechanism was through inhibiting LPS-induced PI3K/AKT/NF-κB signaling pwathway. Plantamajoside may have protective effects against periodontitis.
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Gingival fibroblasts have been known as the major cells for periodontitis [15]. In the development of periodontitis, LPS from Gram-negative bacteria could induce the production of inflammatory mediators [16]. The excess expression of PGE2 and NO following gingival fibroblasts activation contributed to the development of periodontitis [17]. Furthermore, the release of cytokine IL-8 could induce the recruitment of neutrophils, which subsequently release the inflammatory mediators and amplify the inflammatory response [18]. Elevated PGE2, NO, IL-6, and IL-8 were observed in patients of periodontitis [19]. PGE2 was a key inflammatory mediator involved in periodontitis. It is a strong stimulator of bone resorption and formation. It could stimulate collagen synthesis and regulate bone density [20]. NO could stimulate cyclooxygenase and metalloproteinases which may result in periodontal tissue damage. IL-6 was a powerful stimulator of osteoclast differentiation and bone resorption [21]. Therefore, inhibition of these inflammatory mediators could attenuate the damage of the supporting tissues of the teeth and protect periodontitis. In this study, our results showed that plantamajoside significantly reduced the production of these inflammatory mediators. These results indicated that plantamajoside had anti-inflammatory activity against periodontitis and might have protective effects against periodontitis. NF-κB is associated with the expression of inflammatory mediators. Activation of NF-κB could induce the production of inflammatory mediators [22]. Emerging evidence demonstrated that LPS from Porphyromonas gingivalis could induce the activation of NF-κB [23]. And NF-κB has been identified as an important target for the treatment of periodontitis [24]. Furthermore, inhibition of NF-κB activation could attenuate the pathologic process of periodontitis [25]. The present study we found plantamajoside significantly attenuated LPS-induced NF-κB activation. To identify the upstream signaling by which plantamajoside mediated the inhibition of NF-κB activation, PI3K/AKT signaling pathway was measured in this study. Our results showed that plantamajoside significantly attenuated LPS-induced PI3K and AKT phosphorylation. In summary, the results of this study showed that plantamajoside inhibited PI3K/AKT signaling pathway, which subsequently lead to the inhibition of NF-κB activation and inflammatory mediators production. Plantamajoside may be used as a potential candidate for the treatment of periodontitis. Conflict of interest statement
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All authors declare that they have no conflict of interest. References
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Figure Legends Figure 1. Effects of plantamajoside on the cell viability of HGFs. The cell viability was determined by MTT assay. The values presented are the means ± SEM of three independent experiments.
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Figure 2. Plantamajoside inhibits LPS-induced PGE2 and NO production in HGFs. The data presented are the means ± SEM of three independent experiments. #p < 0.05 vs. control group; *p < 0.05, **p < 0.01 vs. LPS group. Figure 3. Plantamajoside inhibits LPS-induced IL-6 and IL-8 production in HGFs. The data presented are the means ± SEM of three independent experiments. #p < 0.05 vs. control group; *p < 0.05, **p < 0.01 vs. LPS group.
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Figure 4. Effects of plantamajoside on LPS-induced NF-κB activation and IκBα degradation. The values presented are the means ± SEM of three independent experiments. #p < 0.05 vs. control group; *p < 0.05, **p < 0.01 vs. LPS group.
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Figure 5. Effects of plantamajoside on LPS-induced PI3K/AKT signaling pathway. The values presented are the means ± SEM of three independent experiments. #p < 0.05 vs. control group; *p < 0.05, **p < 0.01 vs. LPS group.
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Plantamajoside decreased the production of PGE2, NO, IL-6, and IL-8 in LPS-stimulated HGFs. LPS-induced NF-κB p65 and IκB phosphorylation were also suppressed by plantamajoside. Plantamajoside inhibited LPS-induced PI3K and AKT phosphorylation.