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NF-κB pathway
Author’s Accepted Manuscript Pinocembrin inhibits lipopolysaccharide-induced inflammatory mediators production in BV2 microglial cells through suppression of PI3K/Akt/NF-κB pathway Lu-ting Zhou, Ke-jia Wang, Ling Li, Hui Li, Ming Geng www.elsevier.com/locate/ejphar
PII: DOI: Reference:
S0014-2999(15)30064-9 http://dx.doi.org/10.1016/j.ejphar.2015.06.003 EJP70032
To appear in: European Journal of Pharmacology Received date: 15 March 2015 Revised date: 21 May 2015 Accepted date: 1 June 2015 Cite this article as: Lu-ting Zhou, Ke-jia Wang, Ling Li, Hui Li and Ming Geng, Pinocembrin inhibits lipopolysaccharide-induced inflammatory mediators production in BV2 microglial cells through suppression of PI3K/Akt/NF-κB p a t h w a y , European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2015.06.003 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 galley proof before it is published in its final citable 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.
Pinocembrin inhibits lipopolysaccharide-induced inflammatory mediators production in BV2 microglial cells through suppression of PI3K/Akt/NF-κB pathway Lu-ting Zhou1,2, Ke-jia Wang2, Ling Li2, Hui Li2, Ming Geng1 1. Department of Pathology, General Hospital of Jinan Military Command, Jinan, 250031, Shandong Province, China. 2. Department of Pathophysiology, Second Military Medical University, Shanghai, 200433, China. Correspondence to: Ming Geng, MM, Department of Pathology, General Hospital of Jinan Military Command, Jinan, 250031, Shandong Province, China. E-mail: [email protected] Abstract Pinocembrin, one of the primary flavonoids from Pinus heartwood and Eucalyptus, has been reported to have anti-inflammatory and antioxidant activity. This study was designed to evaluate the inhibitory effects of pinocembrin on inflammatory mediators production in LPS-stimulated BV2 microglial cells. The results showed that pinocembrin dose-dependently inhibited LPS-induced inflammatory mediators TNF-α, IL-1β, NO and PGE2 production. Pinocembrin also inhibited LPS-induced iNOS and COX-2 expression. Moreover, pinocembrin inhibited LPS-induced PI3K, Akt phosphorylation, and NF-κB activation, which were required for inflammatory mediators production. Furthermore, treatment of pinocembrin induced nuclear translocation of Nrf2 and expression of HO-1. In conclusion, our data indicated that pinocembrin inhibited LPS-induced inflammatory mediators production by suppressing PI3K/Akt/NF-κB signaling pathway. Keywords: Pinocembrin; LPS; NF-κB; PI3K; microglia 1. Introduction Microglia, the main immune defense cells in the brain, has been reported to play critical roles in immune surveillance under normal conditions (Kim et al., 2000). Microglia activation has been reported to play an important role in the pathogenesis of several neurodegenerative disorders (Liu and Hong, 2003a). In neuroinflammatory pathology, LPS stimulated microglia could induce NF-κB activation and inflammatory mediators such as TNF-α, PGE2 and NO production (Jin et al., 2006). Excessive production of these inflammatory mediators could cause neuronal damage and death (Bazan et al., 1995). Accumulated evidences suggested that inhibition of microglial activation and inflammatory mediators production had the ability to attenuate the severity of neurodegenerative diseases (Dheen et al., 2007; Rock and Peterson, 2006). Therefore, we tried to find compounds from natural products that had the ability to inhibit inflammatory mediators production. Pinocembrin (Fig. 1A), one of the primary flavonoids from Pinus heartwood and Eucalyptus, has been reported to have a variety of pharmacological activities such as anti-inflammatory and antioxidant activities (Saad et al., 2015). Previous studies showed that pinocembrin inhibited LPS-induced TNF-α, IL-1β and IL-6 production in RAW264.7 cells in vitro (Soromou et al., 2012). Pinocembrin also attenuated 6-OHDA-induced neuronal cell death in SH-SY5Y cells (Jin et al., 2014). In vivo, pinocembrin was found to inhibit LPS-induced acute lung injury in mice and inhibit LPS-induced endotoxic shock in mice (Soromou et al., 2014). However, the effect of
pinocembrin on LPS-stimulated BV2 microglial cells remains unclear. Thus, the purpose of this study was to investigate the anti-inflammatory effects and mechanism of pinocembrin on LPS-stimulated BV2 microglial cells. 2. Materials and methods 2.1. Materials Pinocembrin was purchased from National Institute for food and drug control of China (Beijing, China). Griess Reagent was purchased from Beyotime Institute of Biotechnology (Shanghai, China). LPS (Escherichia coli O55:B5) was purchased from Sigma (St. Louis, MO, USA). Antibodies against Nrf2, HO-1, Akt, p-Akt, PI3K, p65, p-p65, IκBα and p-IκBα were obtained from Santa Cruz Biotechnology (Santa Cruz, NYCA, USA). ELISA kits of TNF-α, IL-1β, and PGE2 were purchased from R&D Systems (Minneapolis, MN, USA). 2.2. Cell culture BV2 microglia cells were purchased from the Institute of Basic Medical Sciences of the China Science Academy. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified incubator under 5% CO2. All animal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. 2.3. Cell viability assay A CCK-8 assay was used to measure cell viability. Briefly, BV2 microglia cells were seeded at a density of 5×104 cells/ml in 96 well plates and treated with pinocembrin for 2 h. Then, the cells were stimulated with LPS for 24 h. Thereafter, the cells were incubated with 20 μl of CCK-8 for an additional 2 h. Absorbance was measured at 490 nm using a microplate reader (Sunrise, Tecan). 2.4. ELISA assays BV2 cells were treated with different concentrations of pinocembrin for 1 h and then stimulating by LPS for 24 h. The productions of TNF-α, IL-1β, and PGE2 in the culture supernatant were measured using a commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacture’s protocol. 2.5. Nitrite measurement BV2 cells were pretreated with pinocembrin 1 h and then stimulated by LPS for 24 h. Then 100 μL supernatant was harvested, mixed with 100 μL Griess reagent, and incubated for 15 min at room temperature in the dark. Absorbance at 540 nm of the reaction was monitored with a microplate reader. 2.6. Western blot analysis BV2 cells were pretreated with pinocembrin for 1 h and then stimulated by LPS for 30 min. The cells were lysed with ice-cold lysis buffer supplemented with the protease inhibitor cocktail. Protein concentrations were determined by BCA protein assay kit. Then the proteins were separated on 12% SDS-polyacrylamide gel and transferred onto PVDF membrane. The membranes were blocked with 5% skim milk and incubated with the corresponding primary antibodies. Then the membranes were incubated with secondary antibody coupled to horseradish peroxidase. Then the signals were developed using ECL chemiluminescence reagent (Amersham Pharmacia Biotec, Buckinghamshire, UK). 2.7. Statistical analysis
Data is presented as the mean ± S.E.M. of three independent experiments. Statistical analysis was performed by two-tailed t test or one-way ANOVA. A significant difference was assumed at a level of P < 0.05 or P < 0.01. 3. Results 3.1. Cell toxicity of pinocembrin on BV2 cells To assess the cytotoxic effect of pinocembrin on BV2 cells, the cells were pretreated with different concentrations of pinocembrin and the cell viability was tested by CCK-8. The results showed that pinocembrin at concentrations from 0 to 200 μg/ml had no cytotoxic effect on BV-2 microglial cells (Fig. 1B). 3.2. Pinocembrin suppresses NO and PGE2 production induced by LPS The effects of pinocembrin on inflammatory mediators NO and PGE2 production were detected by Griess reaction and ELISA. The results showed that LPS stimulation remarkably increased the production of NO and PGE2. However, treatment of pinocembrin significantly suppressed LPS-induced NO and PGE2 production (Fig. 2A). 3.3. Pinocembrin inhibits LPS-induced iNOS and COX-2 expression The effects of pinocembrin on LPS-induced iNOS and COX-2 expression were detected by Western blotting. The results showed that LPS significantly increased the expression of iNOS and COX-2. However, treatment of pinocembrin dose-dependently inhibited LPS-induced iNOS and COX-2 expression (Fig. 2B). 3.4. Pinocembrin suppresses TNF-α and IL-1ß production induced by LPS The effects of pinocembrin on inflammatory cytokines TNF-α and IL-1ß production were detected by ELISA. The results showed that LPS stimulation remarkably increased the production of TNF-α and IL-1ß. However, treatment of pinocembrin significantly suppressed LPS-induced TNF-α and IL-1ß production (Fig. 3). 3.5. Effects of pinocembrin on LPS-induced NF-κB activation To determine the effects of pinocembrin on the regulation of NF-κB phosphorylation, the p-NF-κB protein was examined by Western blotting. As shown in Fig. 4, treatment of BV2 cells with LPS alone increased the phosphorylation of NF-κB p65 and IκBα. However, pinocembrin inhibited LPS-induced the phosphorylation of IκB-α and NF-κB p65 (Fig. 4). 3.6. Effects of pinocembrin on LPS-induced PI3K and Akt phosphorylation To further charify the anti-inflammatory mechanism of pinocembrin, the effects of pinocembrin on LPS-induced PI3K and Akt phosphorylation were detected. As shown in Fig. 5, pinocembrin was found to inhibit LPS-induced PI3K and Akt phosphorylation, which are upstream molecules of NF-κB. These results indicated that pinocembrin inhibited LPS-induced NF-κB activation and inflammatory mediators production by suppressing PI3K/Akt signaling pathway. 3.7. Effects of pinocembrin on the expression of Nrf2 and HO-1 The effects of pinocembrin on Nrf2 and HO-1 expression were detected by Western blotting in
this study. Our results showed that LPS up-regulated the expressions of Nrf2 and HO-1. Treatment of pinocembrin augmented the expressions of Nrf2 and HO-1 induced by LPS (Fig. 6). 4. Discussion Pinocembrin, one of the primary flavonoids from Pinus heartwood and Eucalyptus, has been reported to have anti-inflammatory effects (Gao et al., 2010). In this study, our results demonstrated that pinocembrin pretreatment inhibited microglia activation by suppressing inflammatory mediators NO, PGE2, TNF-α, and IL-1β production. The results indicated that pinocembrin had a potential to act as anti-inflammatory agent for treatment of neuroinflammatory diseases. Microglia, the resident macrophages of the central nervous system, has been reported to be the main cells in mediating neuroinflammation (Rock et al., 2004). Activation of microglia by LPS could induce the production of inflammatory mediators NO and PGE2, and inflammatory cytokines TNF-α and IL-1β (Liu et al., 2011; Wang et al., 2004). Previous studies showed that inflammatory mediators NO and PGE2 played a critical role in the pathogenesis of neurodegenerative disorders such as Alzheimer’s disease (Bazan et al., 2002). Overproduction of NO and PGE2 is associated with up-regulation of iNOS and COX-2 (Liu et al., 2008). Inflammatory cytokines TNF-α and IL-1β increased significantly in neurodegenerative disorders (Grammas and Ovase, 2001). Recent studies demonstrated that inhibition the production of inflammatory mediators and cytokines appeared to be beneficial in the treatment of neurodegenerative disorders (Liu and Hong, 2003b). In this study, the results showed that pinocembrin inhibited LPS-induced NO, PGE2, TNF-α, and IL-1β production, as well as iNOS and COX-2 expression. These results suggested that TA exhibited anti-inflammatory and antioxidant effects on LPS-stimulated BV2 microglial cells. It is well known that NF-κB is an important regulator of inflammatory mediators during inflammation (Yu et al., 2013; Zhao et al., 2014). Recent studies showed that NF-κB plays a critical role in microglial activation by regulating of inflammatory mediators NO, PGE2, TNF-α, and IL-1β production (Dang et al., 2014; Velagapudi et al., 2014). Therefore, modulation of NF-κB activation is considered to be a well method to control microglial activation. In the present study, we found that pinocembrin inhibited LPS-induced NF-κB activation in BV2 microglial cells. PI3K and Akt, the upstream molecules of NF-κB, have been demonstrated to play important roles in NF-κB activation (Chen et al., 2014; Qi et al., 2012). In this study, we found that pinocembrin inhibited LPS-induced PI3K/Akt phosphorylation. In addition, previous study suggested that pinocembrin could enter into the cell (Liu et al., 2014). Nrf2, a redox sensitive transcription factor, has been reported to play critical roles in regulating antioxidant and inflammatory responses (Huang et al., 2015). In normal conditions, Nrf2 is sequestered in the cytoplasm and bound with Keap1 (Sid et al., 2014). Activating of Nrf2 could induce the expression of HO-1, which has been demonstrated to have antioxidant activity (Juan et al., 2005; Reisman et al., 2009). Previous report suggested that Nrf2 knockout mice were more hypersensitive to LPS-induced neuroinflammation (Innamorato et al., 2008). In this study, our results demonstrated that treatment of pinocembrin augmented LPS-induced Nrf2 and HO-1 expression. Collectively, these results indicated that the anti-inflammatory and antioxidant effects of pinocembrin may be via inhibition of PI3K/Akt signaling pathway and activation of Nrf2/HO-1 signaling pathway.
In conclusion, our results showed pinocembrin, an extract from Pinus heartwood and Eucalyptus, could inhibit LPS-induced microglial activation. Pinocembrin mediated its effects by suppressing PI3K/Akt/NF-κB signaling pathway. These results indicated that pinocembrin had potential for the treatment of neurodegenerative diseases. Further studies we will investigate the effects of pinocembrin on neuroinflammatory diseases in vivo mice model. Conflict of interest statement All authors declare that they have no conflict of interest. References Bazan, N.G., Colangelo, V., Lukiw, W.J., 2002. Prostaglandins and other lipid mediators in Alzheimer's disease. Prostaglandins & other lipid mediators 68-69, 197-210. Bazan, N.G., Rodriguez de Turco, E.B., Allan, G., 1995. Mediators of injury in neurotrauma: intracellular signal transduction and gene expression. Journal of neurotrauma 12, 791-814. Chen, K.C., Chen, C.Y., Lin, C.J., Yang, T.Y., Chen, T.H., Wu, L.C., Wu, C.C., 2014. Luteolin attenuates TGF-beta 1-induced epithelial-mesenchymal transition of lung cancer cells by interfering in the PI3K/Akt-NF-kappa B-Snail pathway (vol 94, pg 924, 2013). Life Sciences 109, 127-127. Dang, Y.L., Xu, Y.S., Wu, W.T., Li, W.Y., Sun, Y.R., Yang, J., Zhu, Y., Zhang, C., 2014. Tetrandrine Suppresses Lipopolysaccharide-Induced Microglial Activation by Inhibiting NF-kappa B and ERK Signaling Pathways in BV2 Cells. Plos One 9. Dheen, S.T., Kaur, C., Ling, E.A., 2007. Microglial activation and its implications in the brain diseases. Current Medicinal Chemistry 14, 1189-1197. Gao, M., Zhu, S.Y., Tan, C.B., Xu, B., Zhang, W.C., Du, G.H., 2010. Pinocembrin protects the neurovascular unit by reducing inflammation and extracellular proteolysis in MCAO rats. J Asian Nat Prod Res 12, 407-418. Grammas, P., Ovase, R., 2001. Inflammatory factors are elevated in brain microvessels in Alzheimer's disease. Neurobiol Aging 22, 837-842. Huang, C.S., Lin, A.H., Yang, T.C., Liu, K.L., Chen, H.W., Lii, C.K., 2015. Shikonin inhibits oxidized LDL-induced monocyte adhesion by suppressing NF kappa B activation via up-regulation of PI3K/Akt/Nrf2-dependent antioxidation in EA.hy926 endothelial cells. Biochemical pharmacology 93, 352-361. Innamorato, N.G., Rojo, A.I., Garcia-Yague, A.J., Yamamoto, M., de Ceballos, M.L., Cuadrado, A., 2008. The transcription factor Nrf2 is a therapeutic target against brain inflammation. Journal of immunology 181, 680-689. Jin, C.Y., Moon, D.O., Lee, K.J., Kim, M.O., Lee, J.D., Choi, Y.H., Park, Y.M., Kim, G.Y., 2006. Piceatannol attenuates lipopolysaccharide-induced NF-kappaB activation and NF-kappaB-related proinflammatory mediators in BV2 microglia. Pharmacol Res 54, 461-467. Jin, X., Liu, Q., Jia, L., Li, M., Wang, X., 2014. Pinocembrin Attenuates 6-OHDA-induced Neuronal Cell Death Through Nrf2/ARE Pathway in SH-SY5Y Cells. Cellular and molecular neurobiology. Juan, S.H., Cheng, T.H., Lin, H.C., Chu, Y.L., Lee, W.S., 2005. Mechanism of concentration-dependent induction of heme oxygenase-1 by resveratrol in human aortic smooth muscle cells. Biochemical pharmacology 69, 41-48. Kim, W.G., Mohney, R.P., Wilson, B., Jeohn, G.H., Liu, B., Hong, J.S., 2000. Regional difference in susceptibility to lipopolysaccharide-induced neurotoxicity in the rat brain: role of microglia. The
Journal of neuroscience : the official journal of the Society for Neuroscience 20, 6309-6316. Liu, B., Hong, J.S., 2003a. Role of microglia in inflammation-mediated neurodegenerative diseases: mechanisms and strategies for therapeutic intervention. J Pharmacol Exp Ther 304, 1-7. Liu, B., Hong, J.S., 2003b. Role of microglia in inflammation-mediated neurodegenerative diseases: Mechanisms and strategies for therapeutic intervention. J Pharmacol Exp Ther 304, 1-7. Liu, D., Wang, Z., Liu, S., Wang, F., Zhao, S., Hao, A., 2011. Anti-inflammatory effects of fluoxetine in lipopolysaccharide(LPS)-stimulated microglial cells. Neuropharmacology 61, 592-599. Liu, R., Li, J.Z., Song, J.K., Sun, J.L., Li, Y.J., Zhou, S.B., Zhang, T.T., Du, G.H., 2014. Pinocembrin Protects Human Brain Microvascular Endothelial Cells against Fibrillar Amyloid-beta(1-40) Injury by Suppressing the MAPK/NF-kappa B Inflammatory Pathways. BioMed research international. Liu, Z., Fan, Y., Wang, Y., Han, C., Pan, Y., Huang, H., Ye, Y., Luo, L., Yin, Z., 2008. Dipyrithione inhibits lipopolysaccharide-induced iNOS and COX-2 up-regulation in macrophages and protects against endotoxic shock in mice. FEBS letters 582, 1643-1650. Qi, S.M., Xin, Y.Q., Guo, Y.T., Diao, Y., Kou, X.J., Luo, L., Yin, Z.M., 2012. Ampelopsin reduces endotoxic inflammation via repressing ROS-mediated activation of PI3K/Akt/NF-kappa B signaling pathways. Int Immunopharmacol 12, 278-287. Reisman, S.A., Aleksunes, L.M., Klaassen, C.D., 2009. Oleanolic acid activates Nrf2 and protects from acetaminophen hepatotoxicity via Nrf2-dependent and Nrf2-independent processes. Biochemical pharmacology 77, 1273-1282. Rock, R.B., Gekker, G., Hu, S., Sheng, W.S., Cheeran, M., Lokensgard, J.R., Peterson, P.K., 2004. Role of microglia in central nervous system infections. Clin Microbiol Rev 17, 942-964, table of contents. Rock, R.B., Peterson, P.K., 2006. Microglia as a Pharmacological Target in Infectious and Inflammatory Diseases of the Brain. J Neuroimmune Pharm 1, 117-126. Saad, M.A., Abdel Salam, R.M., Kenawy, S.A., Attia, A.S., 2015. Pinocembrin attenuates hippocampal inflammation, oxidative perturbations and apoptosis in a rat model of global cerebral ischemia reperfusion. Pharmacological reports : PR 67, 115-122. Sid, B., Glorieux, C., Valenzuela, M., Rommelaere, G., Najimi, M., Dejeans, N., Renard, P., Verrax, J., Calderon, P.B., 2014. AICAR induces Nrf2 activation by an AMPK-independent mechanism in hepatocarcinoma cells. Biochemical Pharmacology 91, 168-180. Soromou, L.W., Chu, X., Jiang, L.X., Wei, M.M., Huo, M.X., Chen, N., Guan, S., Yang, X.F., Chen, C.Z., Feng, H.H., Deng, X.M., 2012. In vitro and in vivo protection provided by pinocembrin against lipopolysaccharide-induced inflammatory responses. Int Immunopharmacol 14, 66-74. Soromou, L.W., Jiang, L.X., Wei, M.M., Chen, N., Huo, M.X., Chu, X., Zhong, W.T., Wu, Q.C., Balde, A., Deng, X.M., Feng, H.H., 2014. Protection of mice against lipopolysaccharide-induced endotoxic shock by pinocembrin is correlated with regulation of cytokine secretion. J Immunotoxicol 11, 56-61. Velagapudi, R., Aderogba, M., Olajide, O.A., 2014. Tiliroside, a dietary glycosidic flavonoid, inhibits TRAF-6/NF-kappa B/p38-mediated neuroinflammation in activated BV2 microglia. Bba-Gen Subjects 1840, 3311-3319. Wang, T., Qin, L., Liu, B., Liu, Y., Wilson, B., Eling, T.E., Langenbach, R., Taniura, S., Hong, J.S., 2004. Role of reactive oxygen species in LPS-induced production of prostaglandin E2 in microglia. J Neurochem 88, 939-947. Yu, H., Valerio, M., Bielawski, J., 2013. Fenretinide inhibited de novo ceramide synthesis and proinflammatory cytokines induced by Aggregatibacter actinomycetemcomitans. J Lipid Res 54, 189-201.
Zhao, Z.Z., Tang, X.F., Zhao, X.H., Zhang, M.H., Zhang, W.J., Hou, S.H., Yuan, W.F., Zhang, H.F., Shi, L.J., Jia, H., Liang, L., Lai, Z., Gao, J.F., Zhang, K.Y., Fu, L., Chen, W., 2014. Tylvalosin exhibits anti-inflammatory property and attenuates acute lung injury in different models possibly through suppression of NF-kappa B activation. Biochemical Pharmacology 90, 73-87.
Figure Legends Fig. 1 (A) The chemical structure of pinocembrin. (B) Effects of pinocembrin on the cell viability of BV2 microglial cells. Cells were cultured with different concentrations of pinocembrin (50, 100, 200 μg/ml) in the absence or presence of 0.5 μg/mL LPS for 24 h. The cell viability was determined by MTT assay. The values presented are the means ± S.E.M. of three independent experiments. Fig. 2 (A) Effects of pinocembrin on LPS-induced NO and PGE2 production. (B) Effects of pinocembrin on LPS-induced iNOS and COX-2 expression. The data presented are the means ± S.E.M. of three independent experiments. #P < 0.05 vs. control group; *P < 0.05, **P < 0.01 vs. LPS group. Fig. 3 Effects of pinocembrin on LPS-induced TNF-α and IL-1ß production. The data presented are the means ± S.E.M. of three independent experiments. #P < 0.05 vs. control group; *P < 0.05, **P < 0.01 vs. LPS group. Fig. 4 Effects of pinocembrin on LPS-induced NF-κB activation. The values presented are the means ± S.E.M. of three independent experiments. #P< 0.05 vs. control group; *P < 0.05, **P< 0.01 vs. LPS group. Fig. 5 Effects of pinocembrin on LPS-induced PI3K/AKT phosphorylation. The values presented are the means ± S.E.M. of three independent experiments. #P< 0.05 vs. control group; *P < 0.05, **P< 0.01 vs. LPS group. Fig. 6 Effects of pinocembrin on HO-1 and Nrf2 expression. The values presented are the means ± S.E.M. of three independent experiments. #P< 0.05 vs. control group; *P < 0.05, **P< 0.01 vs. LPS group.
PII: DOI: Reference:
S0014-2999(15)30064-9 http://dx.doi.org/10.1016/j.ejphar.2015.06.003 EJP70032
To appear in: European Journal of Pharmacology Received date: 15 March 2015 Revised date: 21 May 2015 Accepted date: 1 June 2015 Cite this article as: Lu-ting Zhou, Ke-jia Wang, Ling Li, Hui Li and Ming Geng, Pinocembrin inhibits lipopolysaccharide-induced inflammatory mediators production in BV2 microglial cells through suppression of PI3K/Akt/NF-κB p a t h w a y , European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2015.06.003 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 galley proof before it is published in its final citable 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.
Pinocembrin inhibits lipopolysaccharide-induced inflammatory mediators production in BV2 microglial cells through suppression of PI3K/Akt/NF-κB pathway Lu-ting Zhou1,2, Ke-jia Wang2, Ling Li2, Hui Li2, Ming Geng1 1. Department of Pathology, General Hospital of Jinan Military Command, Jinan, 250031, Shandong Province, China. 2. Department of Pathophysiology, Second Military Medical University, Shanghai, 200433, China. Correspondence to: Ming Geng, MM, Department of Pathology, General Hospital of Jinan Military Command, Jinan, 250031, Shandong Province, China. E-mail: [email protected] Abstract Pinocembrin, one of the primary flavonoids from Pinus heartwood and Eucalyptus, has been reported to have anti-inflammatory and antioxidant activity. This study was designed to evaluate the inhibitory effects of pinocembrin on inflammatory mediators production in LPS-stimulated BV2 microglial cells. The results showed that pinocembrin dose-dependently inhibited LPS-induced inflammatory mediators TNF-α, IL-1β, NO and PGE2 production. Pinocembrin also inhibited LPS-induced iNOS and COX-2 expression. Moreover, pinocembrin inhibited LPS-induced PI3K, Akt phosphorylation, and NF-κB activation, which were required for inflammatory mediators production. Furthermore, treatment of pinocembrin induced nuclear translocation of Nrf2 and expression of HO-1. In conclusion, our data indicated that pinocembrin inhibited LPS-induced inflammatory mediators production by suppressing PI3K/Akt/NF-κB signaling pathway. Keywords: Pinocembrin; LPS; NF-κB; PI3K; microglia 1. Introduction Microglia, the main immune defense cells in the brain, has been reported to play critical roles in immune surveillance under normal conditions (Kim et al., 2000). Microglia activation has been reported to play an important role in the pathogenesis of several neurodegenerative disorders (Liu and Hong, 2003a). In neuroinflammatory pathology, LPS stimulated microglia could induce NF-κB activation and inflammatory mediators such as TNF-α, PGE2 and NO production (Jin et al., 2006). Excessive production of these inflammatory mediators could cause neuronal damage and death (Bazan et al., 1995). Accumulated evidences suggested that inhibition of microglial activation and inflammatory mediators production had the ability to attenuate the severity of neurodegenerative diseases (Dheen et al., 2007; Rock and Peterson, 2006). Therefore, we tried to find compounds from natural products that had the ability to inhibit inflammatory mediators production. Pinocembrin (Fig. 1A), one of the primary flavonoids from Pinus heartwood and Eucalyptus, has been reported to have a variety of pharmacological activities such as anti-inflammatory and antioxidant activities (Saad et al., 2015). Previous studies showed that pinocembrin inhibited LPS-induced TNF-α, IL-1β and IL-6 production in RAW264.7 cells in vitro (Soromou et al., 2012). Pinocembrin also attenuated 6-OHDA-induced neuronal cell death in SH-SY5Y cells (Jin et al., 2014). In vivo, pinocembrin was found to inhibit LPS-induced acute lung injury in mice and inhibit LPS-induced endotoxic shock in mice (Soromou et al., 2014). However, the effect of
pinocembrin on LPS-stimulated BV2 microglial cells remains unclear. Thus, the purpose of this study was to investigate the anti-inflammatory effects and mechanism of pinocembrin on LPS-stimulated BV2 microglial cells. 2. Materials and methods 2.1. Materials Pinocembrin was purchased from National Institute for food and drug control of China (Beijing, China). Griess Reagent was purchased from Beyotime Institute of Biotechnology (Shanghai, China). LPS (Escherichia coli O55:B5) was purchased from Sigma (St. Louis, MO, USA). Antibodies against Nrf2, HO-1, Akt, p-Akt, PI3K, p65, p-p65, IκBα and p-IκBα were obtained from Santa Cruz Biotechnology (Santa Cruz, NYCA, USA). ELISA kits of TNF-α, IL-1β, and PGE2 were purchased from R&D Systems (Minneapolis, MN, USA). 2.2. Cell culture BV2 microglia cells were purchased from the Institute of Basic Medical Sciences of the China Science Academy. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified incubator under 5% CO2. All animal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. 2.3. Cell viability assay A CCK-8 assay was used to measure cell viability. Briefly, BV2 microglia cells were seeded at a density of 5×104 cells/ml in 96 well plates and treated with pinocembrin for 2 h. Then, the cells were stimulated with LPS for 24 h. Thereafter, the cells were incubated with 20 μl of CCK-8 for an additional 2 h. Absorbance was measured at 490 nm using a microplate reader (Sunrise, Tecan). 2.4. ELISA assays BV2 cells were treated with different concentrations of pinocembrin for 1 h and then stimulating by LPS for 24 h. The productions of TNF-α, IL-1β, and PGE2 in the culture supernatant were measured using a commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacture’s protocol. 2.5. Nitrite measurement BV2 cells were pretreated with pinocembrin 1 h and then stimulated by LPS for 24 h. Then 100 μL supernatant was harvested, mixed with 100 μL Griess reagent, and incubated for 15 min at room temperature in the dark. Absorbance at 540 nm of the reaction was monitored with a microplate reader. 2.6. Western blot analysis BV2 cells were pretreated with pinocembrin for 1 h and then stimulated by LPS for 30 min. The cells were lysed with ice-cold lysis buffer supplemented with the protease inhibitor cocktail. Protein concentrations were determined by BCA protein assay kit. Then the proteins were separated on 12% SDS-polyacrylamide gel and transferred onto PVDF membrane. The membranes were blocked with 5% skim milk and incubated with the corresponding primary antibodies. Then the membranes were incubated with secondary antibody coupled to horseradish peroxidase. Then the signals were developed using ECL chemiluminescence reagent (Amersham Pharmacia Biotec, Buckinghamshire, UK). 2.7. Statistical analysis
Data is presented as the mean ± S.E.M. of three independent experiments. Statistical analysis was performed by two-tailed t test or one-way ANOVA. A significant difference was assumed at a level of P < 0.05 or P < 0.01. 3. Results 3.1. Cell toxicity of pinocembrin on BV2 cells To assess the cytotoxic effect of pinocembrin on BV2 cells, the cells were pretreated with different concentrations of pinocembrin and the cell viability was tested by CCK-8. The results showed that pinocembrin at concentrations from 0 to 200 μg/ml had no cytotoxic effect on BV-2 microglial cells (Fig. 1B). 3.2. Pinocembrin suppresses NO and PGE2 production induced by LPS The effects of pinocembrin on inflammatory mediators NO and PGE2 production were detected by Griess reaction and ELISA. The results showed that LPS stimulation remarkably increased the production of NO and PGE2. However, treatment of pinocembrin significantly suppressed LPS-induced NO and PGE2 production (Fig. 2A). 3.3. Pinocembrin inhibits LPS-induced iNOS and COX-2 expression The effects of pinocembrin on LPS-induced iNOS and COX-2 expression were detected by Western blotting. The results showed that LPS significantly increased the expression of iNOS and COX-2. However, treatment of pinocembrin dose-dependently inhibited LPS-induced iNOS and COX-2 expression (Fig. 2B). 3.4. Pinocembrin suppresses TNF-α and IL-1ß production induced by LPS The effects of pinocembrin on inflammatory cytokines TNF-α and IL-1ß production were detected by ELISA. The results showed that LPS stimulation remarkably increased the production of TNF-α and IL-1ß. However, treatment of pinocembrin significantly suppressed LPS-induced TNF-α and IL-1ß production (Fig. 3). 3.5. Effects of pinocembrin on LPS-induced NF-κB activation To determine the effects of pinocembrin on the regulation of NF-κB phosphorylation, the p-NF-κB protein was examined by Western blotting. As shown in Fig. 4, treatment of BV2 cells with LPS alone increased the phosphorylation of NF-κB p65 and IκBα. However, pinocembrin inhibited LPS-induced the phosphorylation of IκB-α and NF-κB p65 (Fig. 4). 3.6. Effects of pinocembrin on LPS-induced PI3K and Akt phosphorylation To further charify the anti-inflammatory mechanism of pinocembrin, the effects of pinocembrin on LPS-induced PI3K and Akt phosphorylation were detected. As shown in Fig. 5, pinocembrin was found to inhibit LPS-induced PI3K and Akt phosphorylation, which are upstream molecules of NF-κB. These results indicated that pinocembrin inhibited LPS-induced NF-κB activation and inflammatory mediators production by suppressing PI3K/Akt signaling pathway. 3.7. Effects of pinocembrin on the expression of Nrf2 and HO-1 The effects of pinocembrin on Nrf2 and HO-1 expression were detected by Western blotting in
this study. Our results showed that LPS up-regulated the expressions of Nrf2 and HO-1. Treatment of pinocembrin augmented the expressions of Nrf2 and HO-1 induced by LPS (Fig. 6). 4. Discussion Pinocembrin, one of the primary flavonoids from Pinus heartwood and Eucalyptus, has been reported to have anti-inflammatory effects (Gao et al., 2010). In this study, our results demonstrated that pinocembrin pretreatment inhibited microglia activation by suppressing inflammatory mediators NO, PGE2, TNF-α, and IL-1β production. The results indicated that pinocembrin had a potential to act as anti-inflammatory agent for treatment of neuroinflammatory diseases. Microglia, the resident macrophages of the central nervous system, has been reported to be the main cells in mediating neuroinflammation (Rock et al., 2004). Activation of microglia by LPS could induce the production of inflammatory mediators NO and PGE2, and inflammatory cytokines TNF-α and IL-1β (Liu et al., 2011; Wang et al., 2004). Previous studies showed that inflammatory mediators NO and PGE2 played a critical role in the pathogenesis of neurodegenerative disorders such as Alzheimer’s disease (Bazan et al., 2002). Overproduction of NO and PGE2 is associated with up-regulation of iNOS and COX-2 (Liu et al., 2008). Inflammatory cytokines TNF-α and IL-1β increased significantly in neurodegenerative disorders (Grammas and Ovase, 2001). Recent studies demonstrated that inhibition the production of inflammatory mediators and cytokines appeared to be beneficial in the treatment of neurodegenerative disorders (Liu and Hong, 2003b). In this study, the results showed that pinocembrin inhibited LPS-induced NO, PGE2, TNF-α, and IL-1β production, as well as iNOS and COX-2 expression. These results suggested that TA exhibited anti-inflammatory and antioxidant effects on LPS-stimulated BV2 microglial cells. It is well known that NF-κB is an important regulator of inflammatory mediators during inflammation (Yu et al., 2013; Zhao et al., 2014). Recent studies showed that NF-κB plays a critical role in microglial activation by regulating of inflammatory mediators NO, PGE2, TNF-α, and IL-1β production (Dang et al., 2014; Velagapudi et al., 2014). Therefore, modulation of NF-κB activation is considered to be a well method to control microglial activation. In the present study, we found that pinocembrin inhibited LPS-induced NF-κB activation in BV2 microglial cells. PI3K and Akt, the upstream molecules of NF-κB, have been demonstrated to play important roles in NF-κB activation (Chen et al., 2014; Qi et al., 2012). In this study, we found that pinocembrin inhibited LPS-induced PI3K/Akt phosphorylation. In addition, previous study suggested that pinocembrin could enter into the cell (Liu et al., 2014). Nrf2, a redox sensitive transcription factor, has been reported to play critical roles in regulating antioxidant and inflammatory responses (Huang et al., 2015). In normal conditions, Nrf2 is sequestered in the cytoplasm and bound with Keap1 (Sid et al., 2014). Activating of Nrf2 could induce the expression of HO-1, which has been demonstrated to have antioxidant activity (Juan et al., 2005; Reisman et al., 2009). Previous report suggested that Nrf2 knockout mice were more hypersensitive to LPS-induced neuroinflammation (Innamorato et al., 2008). In this study, our results demonstrated that treatment of pinocembrin augmented LPS-induced Nrf2 and HO-1 expression. Collectively, these results indicated that the anti-inflammatory and antioxidant effects of pinocembrin may be via inhibition of PI3K/Akt signaling pathway and activation of Nrf2/HO-1 signaling pathway.
In conclusion, our results showed pinocembrin, an extract from Pinus heartwood and Eucalyptus, could inhibit LPS-induced microglial activation. Pinocembrin mediated its effects by suppressing PI3K/Akt/NF-κB signaling pathway. These results indicated that pinocembrin had potential for the treatment of neurodegenerative diseases. Further studies we will investigate the effects of pinocembrin on neuroinflammatory diseases in vivo mice model. Conflict of interest statement All authors declare that they have no conflict of interest. References Bazan, N.G., Colangelo, V., Lukiw, W.J., 2002. Prostaglandins and other lipid mediators in Alzheimer's disease. Prostaglandins & other lipid mediators 68-69, 197-210. Bazan, N.G., Rodriguez de Turco, E.B., Allan, G., 1995. Mediators of injury in neurotrauma: intracellular signal transduction and gene expression. Journal of neurotrauma 12, 791-814. Chen, K.C., Chen, C.Y., Lin, C.J., Yang, T.Y., Chen, T.H., Wu, L.C., Wu, C.C., 2014. Luteolin attenuates TGF-beta 1-induced epithelial-mesenchymal transition of lung cancer cells by interfering in the PI3K/Akt-NF-kappa B-Snail pathway (vol 94, pg 924, 2013). Life Sciences 109, 127-127. Dang, Y.L., Xu, Y.S., Wu, W.T., Li, W.Y., Sun, Y.R., Yang, J., Zhu, Y., Zhang, C., 2014. Tetrandrine Suppresses Lipopolysaccharide-Induced Microglial Activation by Inhibiting NF-kappa B and ERK Signaling Pathways in BV2 Cells. Plos One 9. Dheen, S.T., Kaur, C., Ling, E.A., 2007. Microglial activation and its implications in the brain diseases. Current Medicinal Chemistry 14, 1189-1197. Gao, M., Zhu, S.Y., Tan, C.B., Xu, B., Zhang, W.C., Du, G.H., 2010. Pinocembrin protects the neurovascular unit by reducing inflammation and extracellular proteolysis in MCAO rats. J Asian Nat Prod Res 12, 407-418. Grammas, P., Ovase, R., 2001. Inflammatory factors are elevated in brain microvessels in Alzheimer's disease. Neurobiol Aging 22, 837-842. Huang, C.S., Lin, A.H., Yang, T.C., Liu, K.L., Chen, H.W., Lii, C.K., 2015. Shikonin inhibits oxidized LDL-induced monocyte adhesion by suppressing NF kappa B activation via up-regulation of PI3K/Akt/Nrf2-dependent antioxidation in EA.hy926 endothelial cells. Biochemical pharmacology 93, 352-361. Innamorato, N.G., Rojo, A.I., Garcia-Yague, A.J., Yamamoto, M., de Ceballos, M.L., Cuadrado, A., 2008. The transcription factor Nrf2 is a therapeutic target against brain inflammation. Journal of immunology 181, 680-689. Jin, C.Y., Moon, D.O., Lee, K.J., Kim, M.O., Lee, J.D., Choi, Y.H., Park, Y.M., Kim, G.Y., 2006. Piceatannol attenuates lipopolysaccharide-induced NF-kappaB activation and NF-kappaB-related proinflammatory mediators in BV2 microglia. Pharmacol Res 54, 461-467. Jin, X., Liu, Q., Jia, L., Li, M., Wang, X., 2014. Pinocembrin Attenuates 6-OHDA-induced Neuronal Cell Death Through Nrf2/ARE Pathway in SH-SY5Y Cells. Cellular and molecular neurobiology. Juan, S.H., Cheng, T.H., Lin, H.C., Chu, Y.L., Lee, W.S., 2005. Mechanism of concentration-dependent induction of heme oxygenase-1 by resveratrol in human aortic smooth muscle cells. Biochemical pharmacology 69, 41-48. Kim, W.G., Mohney, R.P., Wilson, B., Jeohn, G.H., Liu, B., Hong, J.S., 2000. Regional difference in susceptibility to lipopolysaccharide-induced neurotoxicity in the rat brain: role of microglia. The
Journal of neuroscience : the official journal of the Society for Neuroscience 20, 6309-6316. Liu, B., Hong, J.S., 2003a. Role of microglia in inflammation-mediated neurodegenerative diseases: mechanisms and strategies for therapeutic intervention. J Pharmacol Exp Ther 304, 1-7. Liu, B., Hong, J.S., 2003b. Role of microglia in inflammation-mediated neurodegenerative diseases: Mechanisms and strategies for therapeutic intervention. J Pharmacol Exp Ther 304, 1-7. Liu, D., Wang, Z., Liu, S., Wang, F., Zhao, S., Hao, A., 2011. Anti-inflammatory effects of fluoxetine in lipopolysaccharide(LPS)-stimulated microglial cells. Neuropharmacology 61, 592-599. Liu, R., Li, J.Z., Song, J.K., Sun, J.L., Li, Y.J., Zhou, S.B., Zhang, T.T., Du, G.H., 2014. Pinocembrin Protects Human Brain Microvascular Endothelial Cells against Fibrillar Amyloid-beta(1-40) Injury by Suppressing the MAPK/NF-kappa B Inflammatory Pathways. BioMed research international. Liu, Z., Fan, Y., Wang, Y., Han, C., Pan, Y., Huang, H., Ye, Y., Luo, L., Yin, Z., 2008. Dipyrithione inhibits lipopolysaccharide-induced iNOS and COX-2 up-regulation in macrophages and protects against endotoxic shock in mice. FEBS letters 582, 1643-1650. Qi, S.M., Xin, Y.Q., Guo, Y.T., Diao, Y., Kou, X.J., Luo, L., Yin, Z.M., 2012. Ampelopsin reduces endotoxic inflammation via repressing ROS-mediated activation of PI3K/Akt/NF-kappa B signaling pathways. Int Immunopharmacol 12, 278-287. Reisman, S.A., Aleksunes, L.M., Klaassen, C.D., 2009. Oleanolic acid activates Nrf2 and protects from acetaminophen hepatotoxicity via Nrf2-dependent and Nrf2-independent processes. Biochemical pharmacology 77, 1273-1282. Rock, R.B., Gekker, G., Hu, S., Sheng, W.S., Cheeran, M., Lokensgard, J.R., Peterson, P.K., 2004. Role of microglia in central nervous system infections. Clin Microbiol Rev 17, 942-964, table of contents. Rock, R.B., Peterson, P.K., 2006. Microglia as a Pharmacological Target in Infectious and Inflammatory Diseases of the Brain. J Neuroimmune Pharm 1, 117-126. Saad, M.A., Abdel Salam, R.M., Kenawy, S.A., Attia, A.S., 2015. Pinocembrin attenuates hippocampal inflammation, oxidative perturbations and apoptosis in a rat model of global cerebral ischemia reperfusion. Pharmacological reports : PR 67, 115-122. Sid, B., Glorieux, C., Valenzuela, M., Rommelaere, G., Najimi, M., Dejeans, N., Renard, P., Verrax, J., Calderon, P.B., 2014. AICAR induces Nrf2 activation by an AMPK-independent mechanism in hepatocarcinoma cells. Biochemical Pharmacology 91, 168-180. Soromou, L.W., Chu, X., Jiang, L.X., Wei, M.M., Huo, M.X., Chen, N., Guan, S., Yang, X.F., Chen, C.Z., Feng, H.H., Deng, X.M., 2012. In vitro and in vivo protection provided by pinocembrin against lipopolysaccharide-induced inflammatory responses. Int Immunopharmacol 14, 66-74. Soromou, L.W., Jiang, L.X., Wei, M.M., Chen, N., Huo, M.X., Chu, X., Zhong, W.T., Wu, Q.C., Balde, A., Deng, X.M., Feng, H.H., 2014. Protection of mice against lipopolysaccharide-induced endotoxic shock by pinocembrin is correlated with regulation of cytokine secretion. J Immunotoxicol 11, 56-61. Velagapudi, R., Aderogba, M., Olajide, O.A., 2014. Tiliroside, a dietary glycosidic flavonoid, inhibits TRAF-6/NF-kappa B/p38-mediated neuroinflammation in activated BV2 microglia. Bba-Gen Subjects 1840, 3311-3319. Wang, T., Qin, L., Liu, B., Liu, Y., Wilson, B., Eling, T.E., Langenbach, R., Taniura, S., Hong, J.S., 2004. Role of reactive oxygen species in LPS-induced production of prostaglandin E2 in microglia. J Neurochem 88, 939-947. Yu, H., Valerio, M., Bielawski, J., 2013. Fenretinide inhibited de novo ceramide synthesis and proinflammatory cytokines induced by Aggregatibacter actinomycetemcomitans. J Lipid Res 54, 189-201.
Zhao, Z.Z., Tang, X.F., Zhao, X.H., Zhang, M.H., Zhang, W.J., Hou, S.H., Yuan, W.F., Zhang, H.F., Shi, L.J., Jia, H., Liang, L., Lai, Z., Gao, J.F., Zhang, K.Y., Fu, L., Chen, W., 2014. Tylvalosin exhibits anti-inflammatory property and attenuates acute lung injury in different models possibly through suppression of NF-kappa B activation. Biochemical Pharmacology 90, 73-87.
Figure Legends Fig. 1 (A) The chemical structure of pinocembrin. (B) Effects of pinocembrin on the cell viability of BV2 microglial cells. Cells were cultured with different concentrations of pinocembrin (50, 100, 200 μg/ml) in the absence or presence of 0.5 μg/mL LPS for 24 h. The cell viability was determined by MTT assay. The values presented are the means ± S.E.M. of three independent experiments. Fig. 2 (A) Effects of pinocembrin on LPS-induced NO and PGE2 production. (B) Effects of pinocembrin on LPS-induced iNOS and COX-2 expression. The data presented are the means ± S.E.M. of three independent experiments. #P < 0.05 vs. control group; *P < 0.05, **P < 0.01 vs. LPS group. Fig. 3 Effects of pinocembrin on LPS-induced TNF-α and IL-1ß production. The data presented are the means ± S.E.M. of three independent experiments. #P < 0.05 vs. control group; *P < 0.05, **P < 0.01 vs. LPS group. Fig. 4 Effects of pinocembrin on LPS-induced NF-κB activation. The values presented are the means ± S.E.M. of three independent experiments. #P< 0.05 vs. control group; *P < 0.05, **P< 0.01 vs. LPS group. Fig. 5 Effects of pinocembrin on LPS-induced PI3K/AKT phosphorylation. The values presented are the means ± S.E.M. of three independent experiments. #P< 0.05 vs. control group; *P < 0.05, **P< 0.01 vs. LPS group. Fig. 6 Effects of pinocembrin on HO-1 and Nrf2 expression. The values presented are the means ± S.E.M. of three independent experiments. #P< 0.05 vs. control group; *P < 0.05, **P< 0.01 vs. LPS group.