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Anti-inflammatory effects of farrerol on IL-1β-stimulated human osteoarthritis chondrocytes
European Journal of Pharmacology 764 (2015) 443–447
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Anti-inflammatory effects of farrerol on IL-1β-stimulated human osteoarthritis chondrocytes Hongfei Zhang n, Jiapeng Yan, Yuesheng Zhuang, Guiquan Han Department of Orthopedics, Affiliated Hospital of Weifang Medical University, Weifang, Shandong 261031, China
art ic l e i nf o
a b s t r a c t
Article history: Received 3 June 2015 Received in revised form 3 July 2015 Accepted 6 July 2015 Available online 7 July 2015
The present study aimed to investigate the anti-inflammatory effects and the underlying molecular mechanism of farrerol on IL-1β-stimulated human osteoarthritis chondrocytes. Chondrocytes were pretreated with farrerol 1 h before IL-1β stimulation. The effects of farrerol on NO and PGE2 production were tested by Griess reagent and ELISA. The effects of farrerol on COX-2, iNOS, Akt, phosphorylated Akt, and NF-κB activation were measured by western blot analysis. The results showed that farrerol remarkably inhibited IL-1β-induced NO and PGE2 production, as well as COX-2 and iNOS expression. Farrerol also inhibited IL-1β-induced NF-κB activation. Furthermore, farrerol significantly inhibited IL1β-induced phosphorylation of PI3K and Akt. In conclusion, these results indicated that farrerol inhibited IL-1β-induced inflammatory responses in osteoarthritis chondrocytes by blocking PI3K/Akt/NF-κB signaling pathway. & 2015 Elsevier B.V. All rights reserved.
Keywords: Farrerol Osteoarthritis chondrocyte IL-1β NF-κB PI3K
1. Introduction Osteoarthritis (OA) is one of the most prevalent types of arthritis that is characterized by articular cartilage degradation and destruction of cartilage matrix (Goldring and Goldring, 2004). Chondrocyte has been reported to play critical roles in the development of OA (Blanco et al., 1998). IL-1β could induce NF-κB activation which subsequently induced the production of inflammatory mediators such as NO and PGE2 in osteoarthritis chondrocytes (Largo et al., 2003). These inflammatory mediators amplify the inflammation and lead to chronic pain and the pathophysiology of OA (Miller et al., 2009). Accumulated evidences suggested that anti-inflammatory agents capable of inhibiting the production of inflammatory mediators may have the potential to treat OA (Rashad et al., 1989). Many herbal products have been reported to have anti-inflammatory effects and it is of clinical interest to seek herbal products to treat OA (Hu et al., 2011; Liu et al., 2013). Farrerol, a new kind of 2,3-dihydro-flavonoid isolated from rhododendron, has been reported to have various pharmacological activities, such as anti-inflammatory, anti-bacterial, and antioxidant effects (Qiu et al., 2011; Yang et al., 2013). Previous studies showed that farrerol had protective effects against hydrogen-peroxide-induced apoptosis in human endothelium-derived EA.hy926 n
Corresponding author. E-mail address: [email protected] (H. Zhang).
http://dx.doi.org/10.1016/j.ejphar.2015.07.012 0014-2999/& 2015 Elsevier B.V. All rights reserved.
cells (Li et al., 2013). Farrerol also suppressed murine T lymphocyte activation both in vitro and in vivo (Xiong et al., 2013). In addition, farrerol has been reported to attenuate LPS-induced acute lung injury in mice (Ci et al., 2012). However, the protective effect of farrerol on osteoarthritis has been reported. In the present study, we investigated the anti-inflammatory effect and mechanism of farrerol in IL-1β-stimulated human osteoarthritis chondrocytes.
2. Materials and methods 2.1. Chemicals and reagents Recombinant human IL-1β, ELISA kit for PGE2 was purchased from R&D systems (Minneapolis, MN, USA). Antibodies against PI3K, AKT, p-AKT, COX-2, iNOS, p65, p-p65, IκBα, and p-IκBα were purchased from Cell Signaling Technology Inc. (Beverly, MA). 3-(4,5dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) and farrerol were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Griess Reagent was purchased from Beyotime Institute of Biotechnology (Shanghai, China). 2.2. Cell culture The experiment was in accordance with the Declaration of Helsinki and Tokyo. Articular cartilage samples were obtained from 20 patients (age: 627 8) undergoing total knee replacement
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surgery. Primary chondrocytes were isolated from articular cartilage as described previously (Cheng et al., 2011). In brief, cartilage were digested with 0.25% trypsin for 30 min and further digested by 2 mg/ml collagenase II in DMEM for 6 h at 37 °C. Then the cells were resuspended in DMEM containing 10% fetal bovine serum (FBS) and cultured at 37 °C with 5% CO2. Cells between passages 1 and 3 were used in this study. 2.3. MTT assay Cell viability was assessed by using MTT assay. In brief, chondrocytes were seeded in a 96-well plate and cultured for 12 h. Then the cells were treated with different concentrations of farrerol for 1 h and stimulated with IL-1β (10 ng/ml) for 24 h. Subsequently, the cells were incubated with MTT (5 mg/ml) at 37 °C for 4 h. The insoluble formazan product was dissolved in DMSO. Absorbance at 490 nm was measured using a Dynatech MR5000 plate reader (Dynatech, Chantilly, VA, USA). 2.4. ELISA assay The effects of farrerol on IL-1β-induced PGE2 production were detected by ELISA. Chondrocytes were pretreated with farrerol 1 h and then stimulated with IL-1β (10 ng/ml) for 24 h. The level of PGE2 in the culture medium was quantified using a specific ELISA kit (R&D Systems, Minneapoils, MN) according to the manufacturer's instructions.
Fig. 1. Effects of farrerol on the cell viability of chondrocytes. Cells were cultured with different concentrations of farrerol (0–60 μM) for 24 h. The cell viability was determined by MTT assay. The values presented are the means 7 S.E.M. of three independent experiments. *Po 0.05, **Po 0.01 vs. control group.
2.5. Quantification of NO The effects of farrerol on IL-1β-induced NO production were detected by Griess reagent. Chondrocytes were pretreated with farrerol 1 h and then stimulated with IL-1β (10 ng/ml) for 24 h. The concentration of NO in the culture medium was tested using the Griess reagent according to the manufacturer’s instructions. 2.6. Western blot analysis Total proteins from chondrocytes were extracted by M-PER Mammalian Protein Extraction Reagent (Thermo). Protein concentration was determined using the BCA method. 40 μg protein for each sample was separated on 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% nonfat dry milk and incubated with primary antibodies NF-κB p65, IκBα, p-IκBα, COX-2, iNOS, PI3K, AKT, p-AKT at 4 °C overnight. Then the membranes were incubated with the HRP-conjugated secondary antibodies at room temperature for 2 h. The signals on the membrane were measured using the ECL detection reagents (Thermo). The bands were quantified using the Quantity One software package (Bio-Rad Laboratories, UK). 2.7. Statistical analysis Data are presented as the mean 7S.E.M. Statistical significance of different groups were performed by one-way analysis of variance followed by Dunnett's test. P o0.05 were considered to indicate statistical significance.
3. Results 3.1. Effects of farrerol on cell viability Cell viability of chondrocytes was assessed by using MTT assay. As shown in Fig. 1, no cytotoxicity of farrerol on chondrocytes was observed at the doses of 0–50 μM. The results showed a reduction
Fig. 2. Farrerol inhibits IL-1β-induced NO and PGE2 production. Chondrocytes were pretreated with farrerol 1 h and then stimulated with IL-1β (10ng/ml) for 24 h. The levels of NO and PGE2 were detected. The data presented are the means 7 S.E.M. of three independent experiments. #P o 0.05 vs. control group; *Po 0.05, **Po 0.01 vs. IL-1β group.
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of IL-1β-induced cell viability compared with the control group. Farrerol at doses of 25 and 50 μM reversed the effects of IL-1β on cell viability. Thus, farrerol at the doses of 12.5, 25, 50 μM were used in the subsequent studies. 3.2. Effects of farrerol on IL-1β-induced NO and PGE2 production To investigate the anti-inflammatory effects of farrerol, the effects of farrerol on IL-1β-induced NO and PGE2 production were measured in this study. As shown in Fig. 2, IL-1β significantly upregulated the production of NO and PGE2. However, farrerol dosedependently suppressed IL-1β-induced NO and PGE2 production.
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3.3. Effects of farrerol on IL-1β-induced iNOS and COX-2 expression In this study, the effects of farrerol on IL-1β-induced iNOS and COX-2 expression were detected by western blot analysis. The results showed that compared to the control group, IL-1β remarkably up-regulated the expression of iNOS and COX-2. However, farrerol dose-dependently suppressed IL-1β-induced iNOS and COX-2 expression (Fig. 3). 3.4. Farrerol inhibits IL-1β-induced NF-κB activation It is well known that NF-κB plays an important role in the regulation of inflammatory mediators. To investigate the anti-
Fig. 3. Farrerol inhibits IL-1β-induced iNOS and COX-2 expression. Chondrocytes were pretreated with farrerol 1 h and then stimulated with IL-1β (10 ng/ml) for 24 h. The expression of iNOs and COX-2 were detected by western blot analysis. The data presented are the means 7 S.E.M. of three independent experiments. #P o0.05 vs. control group; *Po 0.05, **P o0.01 vs. IL-1β group.
Fig. 4. Farrerol inhibits IL-1β-induced NF-κB activation and IκBα degradation. Chondrocytes were pretreated with farrerol 1 h and then stimulated with IL-1β (10 ng/ml) for 1 h. NF-κB activation and IκBα degradation were detected by western blot analysis. The values presented are the means 7S.E.M. of three independent experiments. #Po 0.05 vs. control group; *P o0.05, **Po 0.01 vs. IL-1β group.
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inflammatory mechanism of farrerol, the effects of farrerol on IL1β-induced NF-κB activation were detected. The results demonstrated that IL-1β significantly up-regulated NF-κB phosphorylation and IκBα degradation in chondrocytes. And the up-regulation was remarkably inhibited by treatment of farrerol (Fig. 4). 3.5. Farrerol inhibits IL-1β-induced PI3K/Akt phosphorylation To further investigate the anti-inflammatory mechanism of farrerol, the effects of farrerol on IL-1β-induced PI3K/Akt phosphorylation were detected by western blot analysis. As shown in Fig. 5, IL-1β significantly up-regulated the phosphorylation of PI3K and AKT compared to the control group. Treatment of farrerol PI3K/Akt dose-dependently suppressed IL-1β-induced phosphorylation.
4. Discussion Farrerol, a new kind of 2,3-dihydro-flavonoid isolated from rhododendron, has been reported to have therapeutic effects on LPS-induced acute lung injury (Ci et al., 2012). However, the antiinflammatory effects of farrerol on IL-1β-induced inflammation in chondrocytes remain unclear. The present study found that pretreatment with farrerol significantly inhibited IL-1β-induced NO
and PGE2 production, as well as COX-2 and iNOS expression. The anti-inflammatory mechanism of farrerol was mediated via inhibiting IL-1β-induced PI3K and AKT phosphorylation. Osteoarthritis (OA) is a widespread musculoskeletal disease that is characterized by articular cartilage degradation and destruction of cartilage matrix (Loeser, 2009). Pro-inflammatory cytokines such as IL-1β produced by activated chondrocytes are believed to play critical roles in the initiation and development of OA (Moos et al., 2000). Recent studies demonstrated that many natural products had the ability to control or treatment of OA by inhibiting inflammatory mediators production (Ahmed et al., 2005; Shakibaei et al., 2007). In the present study, we found that farrerol inhibited IL-1β-induced NO and PGE2 production in a dose-dependent manner. Next, we investigated whether the inhibitory effect of farrerol on NO and PGE2 production were related to iNOS and COX-2 expression. Our results demonstrated that farrerol inhibited IL-1β-induced NO and PGE2 production by suppressing upstream molecules iNOS and COX-2 expression. In addition, farrerol was found to suppress ConA-induced Th1 and Th2 cytokine production mouse T lymphocytes (Xiong et al., 2013). Farrerol also inhibited LPS-induced TNF-α, IL-6 and IL-8 production (Ci et al., 2012). These studies indicated that farrerol had antiinflammatory effects. Taken together, these results suggested that farrerol had anti-inflammatory effects on IL-1β-induced inflammation in chondrocytes.
Fig. 5. Farrerol inhibits IL-1β-induced PI3K/Akt phosphorylation. Chondrocytes were pretreated with farrerol 1 h and then stimulated with IL-1β (10 ng/ml) for 1 h. PI3K and Akt phosphorylation were detected by western blot analysis. The values presented are the means 7S.E.M. of three independent experiments. #Po 0.05 vs. control group; *Po 0.05, **P o 0.01 vs. IL-1β group.
H. Zhang et al. / European Journal of Pharmacology 764 (2015) 443–447
NF-κB, an important transcriptional factor, has been reported to play critical roles in inflammatory cytokines production (Blackwell and Christman, 1997; Rai et al., 2014). PI3K and Akt, the upstream molecules of NF-κB, have been demonstrated to play important roles in NF-κB activation (Rahman et al., 2004; Wang et al., 2014). Thus, to investigate the anti-inflammatory mechanisms of farrerol, we detected the effects farrerol on PI3K and Akt phosphorylation and NF-κB activation. The results showed that farrerol inhibited IL-1β-induced inflammation in chondrocytes via inhibition of PI3K/Akt phosphorylation and NF-κB activation. MAPK pathways also play critical roles in the regulation of inflammatory cytokines production (Pawate et al., 2004). Recent studies showed that farrerol inhibited hydrogen peroxide-induced ERK1/2 activation in EA.hy926 cells (Li et al., 2014). Farrerol also inhibited hydrogen peroxide-induced p38 MAPK activation in EA. hy926 cells (Li et al., 2013). It has been reported that there are multiple cross-talk points between PI3K and MAPKs pathways (Aksamitiene et al., 2012). Furthermore, AKT activation has been shown to activate MAPK pathway (Binion et al., 2009). In this study, our results showed that farrerol could inhibit IL-1β-induced PI3K and AKT activation. These results suggested that farrerol might have the ability to inhibit MAPK pathways. Our results were consistent with previous studies. In conclusion, our findings showed that farrerol suppressed IL1β-induced production of NO and PGE2, and expression of iNOS and COX-2 in chondrocytes. Furthermore, our molecular data suggest that farrerol exhibited its anti-inflammatory effects by preventing IL-1β-induced PI3K/Akt phosphorylation.
Conflict of interest statement All authors declare that they have no conflict of interest.
References Ahmed, S., Wang, N.Z., Bin Hafeez, B., Cheruvu, V.K., Haqqi, T.M., 2005. Punica granatum L. extract inhibits IL-1 beta-induced expression of matrix metalloproteinases by inhibiting the activation of MAP kinases and NF-kappa B in human chondrocytes in vitro. J. Nutr. 135, 2096–2102. Aksamitiene, E., Kiyatkin, A., Kholodenko, B.N., 2012. Cross-talk between mitogenic Ras/MAPK and survival PI3K/Akt pathways: a fine balance. Biochem. Soc. Trans. 40, 139–146. Binion, D.G., Heidemann, J., Li, M.S., Nelson, V.M., Otterson, M.F., Rafiee, P., 2009. Vascular cell adhesion molecule-1 expression in human intestinal microvascular endothelial cells is regulated by PI 3-kinase/Akt/MAPK/NF-kappa B: inhibitory role of curcumin. Am. J. Physiol. Gastrointest. Liver Physiol. 297, G259–G268. Blackwell, T.S., Christman, J.W., 1997. The role of nuclear factor-kappa B in cytokine gene regulation. Am. J. Respir. Cell Mol. Biol. 17, 3–9. Blanco, F.J., Guitian, R., Vazquez-Martul, E., de Toro, F.J., Galdo, F., 1998. Osteoarthritis chondrocytes die by apoptosis. A possible pathway for osteoarthritis pathology. Arthritis Rheum. 41, 284–289.
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Cheng, A.W., Stabler, T.V., Bolognesi, M., Kraus, V.B., 2011. Selenomethionine inhibits IL-1beta inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2) expression in primary human chondrocytes. Osteoarthritis Cartilage 19, 118–125. Ci, X., Chu, X., Wei, M., Yang, X., Cai, Q., Deng, X., 2012. Different effects of farrerol on an OVA-induced allergic asthma and LPS-induced acute lung injury. PloS One 7, e34634. Goldring, S.R., Goldring, M.B., 2004. The role of cytokines in cartilage matrix degeneration in osteoarthritis. Clin. Orthop. Relat. Res. 427, S27–S36. Hu, P.F., Chen, W.P., Tang, J.L., Bao, J.P., Wu, L.D., 2011. Protective effects of berberine in an experimental rat osteoarthritis model. Phytother. Res. 25, 878–885. Largo, R., Alvarez-Soria, M.A., Diez-Ortego, I., Calvo, E., Sanchez-Pernaute, O., Egido, J., Herrero-Beaumont, G., 2003. Glucosamine inhibits IL-1beta-induced NFkappaB activation in human osteoarthritic chondrocytes. Osteoarthritis Cartilage 11, 290–298. Li, J., Ge, R., Zhao, C., Tang, L., Li, J., Li, Q., 2014. Farrerol regulates occludin expression in hydrogen peroxide-induced EA.hy926 cells by modulating ERK1/2 activity. Eur. J. Pharmacol. 734, 9–14. Li, J.K., Ge, R., Tang, L., Li, Q.S., 2013. Protective effects of farrerol against hydrogenperoxide-induced apoptosis in human endothelium-derived EA.hy926 cells. Can. J. Physiol. Pharmacol. 91, 733–740. Liu, J., Pan, J., Wang, Y., Lin, D., Shen, D., Yang, H., Li, X., Luo, M., Cao, X., 2013. Component analysis of Chinese medicine and advances in fuming-washing therapy for knee osteoarthritis via unsupervised data mining methods. J. Tradit. Chin. Med.¼ Chung i tsa chih ying wen pan 33, 686–691. Loeser, R.F., 2009. Aging and osteoarthritis: the role of chondrocyte senescence and aging changes in the cartilage matrix. Osteoarthritis Cartilage 17, 971–979. Miller, A.H., Maletic, V., Raison, C.L., 2009. Inflammation and its discontents: the role of cytokines in the pathophysiology of major depression. Biol. Psychiatry 65, 732–741. Moos, V., Rudwaleit, M., Herzog, V., Hohlig, K., Sieper, J., Muller, B., 2000. Association of genotypes affecting the expression of interleukin-1beta or interleukin-1 receptor antagonist with osteoarthritis. Arthritis Rheum. 43, 2417–2422. Pawate, S., Shen, Q., Fan, F., Bhat, N.R., 2004. Redox regulation of glial inflammatory response to lipopolysaccharide and interferongamma. J. Neurosci. Res. 77, 540–551. Qiu, J., Xiang, H., Hu, C., Wang, Q., Dong, J., Li, H., Luo, M., Wang, J., Deng, X., 2011. Subinhibitory concentrations of farrerol reduce alpha-toxin expression in Staphylococcus aureus. FEMS Microbiol. Lett. 315, 129–133. Rahman, K.M., Li, Y., Sarkar, F.H., 2004. Inactivation of akt and NF-kappaB play important roles during indole-3-carbinol-induced apoptosis in breast cancer cells. Nutr. Cancer 48, 84–94. Rai, A., Kapoor, S., Singh, S., Chatterji, B.P., Panda, D., 2014. Transcription factor NFkappaB associates with microtubules and stimulates apoptosis in response to suppression of microtubule dynamics in MCF-7 cells. Biochem. Pharmacol. 93, 277–289. Rashad, S., Low, F., Revell, P., Hemingway, A., Rainsford, K., Walker, F., 1989. Effect of non-steroidal anti-inflammatory drugs on course of osteoarthritis. Lancet 2, 1149. Shakibaei, M., John, T., Seifarth, C., Mobasheri, A., 2007. Resveratrol inhibits IL-1 beta-induced stimulation of caspase-3 and cleavage of PARP in human articular chondrocytes in vitro. Ann. N. Y. Acad. Sci. 1095, 554–563. Wang, L., Xu, Y., Yu, Q., Sun, Q., Xu, Y., Gu, Q., Xu, X., 2014. H-RN, a novel antiangiogenic peptide derived from hepatocyte growth factor inhibits inflammation in vitro and in vivo through PI3K/AKT/IKK/NF-kappaB signal pathway. Biochem. Pharmacol. 89, 255–265. Xiong, Y., Zhang, S., Lu, J., Sun, S., Song, B., Xu, L., Yang, Z., Guan, S., 2013. Investigation of effects of farrerol on suppression of murine T lymphocyte activation in vitro and in vivo. Int. Immunopharmacol. 16, 313–321. Yang, Z., Fu, Y., Liu, B., Zhou, E., Liu, Z., Song, X., Li, D., Zhang, N., 2013. Farrerol regulates antimicrobial peptide expression and reduces Staphylococcus aureus internalization into bovine mammary epithelial cells. Microb. Pathog. 65, 1–6.
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Anti-inflammatory effects of farrerol on IL-1β-stimulated human osteoarthritis chondrocytes Hongfei Zhang n, Jiapeng Yan, Yuesheng Zhuang, Guiquan Han Department of Orthopedics, Affiliated Hospital of Weifang Medical University, Weifang, Shandong 261031, China
art ic l e i nf o
a b s t r a c t
Article history: Received 3 June 2015 Received in revised form 3 July 2015 Accepted 6 July 2015 Available online 7 July 2015
The present study aimed to investigate the anti-inflammatory effects and the underlying molecular mechanism of farrerol on IL-1β-stimulated human osteoarthritis chondrocytes. Chondrocytes were pretreated with farrerol 1 h before IL-1β stimulation. The effects of farrerol on NO and PGE2 production were tested by Griess reagent and ELISA. The effects of farrerol on COX-2, iNOS, Akt, phosphorylated Akt, and NF-κB activation were measured by western blot analysis. The results showed that farrerol remarkably inhibited IL-1β-induced NO and PGE2 production, as well as COX-2 and iNOS expression. Farrerol also inhibited IL-1β-induced NF-κB activation. Furthermore, farrerol significantly inhibited IL1β-induced phosphorylation of PI3K and Akt. In conclusion, these results indicated that farrerol inhibited IL-1β-induced inflammatory responses in osteoarthritis chondrocytes by blocking PI3K/Akt/NF-κB signaling pathway. & 2015 Elsevier B.V. All rights reserved.
Keywords: Farrerol Osteoarthritis chondrocyte IL-1β NF-κB PI3K
1. Introduction Osteoarthritis (OA) is one of the most prevalent types of arthritis that is characterized by articular cartilage degradation and destruction of cartilage matrix (Goldring and Goldring, 2004). Chondrocyte has been reported to play critical roles in the development of OA (Blanco et al., 1998). IL-1β could induce NF-κB activation which subsequently induced the production of inflammatory mediators such as NO and PGE2 in osteoarthritis chondrocytes (Largo et al., 2003). These inflammatory mediators amplify the inflammation and lead to chronic pain and the pathophysiology of OA (Miller et al., 2009). Accumulated evidences suggested that anti-inflammatory agents capable of inhibiting the production of inflammatory mediators may have the potential to treat OA (Rashad et al., 1989). Many herbal products have been reported to have anti-inflammatory effects and it is of clinical interest to seek herbal products to treat OA (Hu et al., 2011; Liu et al., 2013). Farrerol, a new kind of 2,3-dihydro-flavonoid isolated from rhododendron, has been reported to have various pharmacological activities, such as anti-inflammatory, anti-bacterial, and antioxidant effects (Qiu et al., 2011; Yang et al., 2013). Previous studies showed that farrerol had protective effects against hydrogen-peroxide-induced apoptosis in human endothelium-derived EA.hy926 n
Corresponding author. E-mail address: [email protected] (H. Zhang).
http://dx.doi.org/10.1016/j.ejphar.2015.07.012 0014-2999/& 2015 Elsevier B.V. All rights reserved.
cells (Li et al., 2013). Farrerol also suppressed murine T lymphocyte activation both in vitro and in vivo (Xiong et al., 2013). In addition, farrerol has been reported to attenuate LPS-induced acute lung injury in mice (Ci et al., 2012). However, the protective effect of farrerol on osteoarthritis has been reported. In the present study, we investigated the anti-inflammatory effect and mechanism of farrerol in IL-1β-stimulated human osteoarthritis chondrocytes.
2. Materials and methods 2.1. Chemicals and reagents Recombinant human IL-1β, ELISA kit for PGE2 was purchased from R&D systems (Minneapolis, MN, USA). Antibodies against PI3K, AKT, p-AKT, COX-2, iNOS, p65, p-p65, IκBα, and p-IκBα were purchased from Cell Signaling Technology Inc. (Beverly, MA). 3-(4,5dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) and farrerol were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Griess Reagent was purchased from Beyotime Institute of Biotechnology (Shanghai, China). 2.2. Cell culture The experiment was in accordance with the Declaration of Helsinki and Tokyo. Articular cartilage samples were obtained from 20 patients (age: 627 8) undergoing total knee replacement
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surgery. Primary chondrocytes were isolated from articular cartilage as described previously (Cheng et al., 2011). In brief, cartilage were digested with 0.25% trypsin for 30 min and further digested by 2 mg/ml collagenase II in DMEM for 6 h at 37 °C. Then the cells were resuspended in DMEM containing 10% fetal bovine serum (FBS) and cultured at 37 °C with 5% CO2. Cells between passages 1 and 3 were used in this study. 2.3. MTT assay Cell viability was assessed by using MTT assay. In brief, chondrocytes were seeded in a 96-well plate and cultured for 12 h. Then the cells were treated with different concentrations of farrerol for 1 h and stimulated with IL-1β (10 ng/ml) for 24 h. Subsequently, the cells were incubated with MTT (5 mg/ml) at 37 °C for 4 h. The insoluble formazan product was dissolved in DMSO. Absorbance at 490 nm was measured using a Dynatech MR5000 plate reader (Dynatech, Chantilly, VA, USA). 2.4. ELISA assay The effects of farrerol on IL-1β-induced PGE2 production were detected by ELISA. Chondrocytes were pretreated with farrerol 1 h and then stimulated with IL-1β (10 ng/ml) for 24 h. The level of PGE2 in the culture medium was quantified using a specific ELISA kit (R&D Systems, Minneapoils, MN) according to the manufacturer's instructions.
Fig. 1. Effects of farrerol on the cell viability of chondrocytes. Cells were cultured with different concentrations of farrerol (0–60 μM) for 24 h. The cell viability was determined by MTT assay. The values presented are the means 7 S.E.M. of three independent experiments. *Po 0.05, **Po 0.01 vs. control group.
2.5. Quantification of NO The effects of farrerol on IL-1β-induced NO production were detected by Griess reagent. Chondrocytes were pretreated with farrerol 1 h and then stimulated with IL-1β (10 ng/ml) for 24 h. The concentration of NO in the culture medium was tested using the Griess reagent according to the manufacturer’s instructions. 2.6. Western blot analysis Total proteins from chondrocytes were extracted by M-PER Mammalian Protein Extraction Reagent (Thermo). Protein concentration was determined using the BCA method. 40 μg protein for each sample was separated on 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% nonfat dry milk and incubated with primary antibodies NF-κB p65, IκBα, p-IκBα, COX-2, iNOS, PI3K, AKT, p-AKT at 4 °C overnight. Then the membranes were incubated with the HRP-conjugated secondary antibodies at room temperature for 2 h. The signals on the membrane were measured using the ECL detection reagents (Thermo). The bands were quantified using the Quantity One software package (Bio-Rad Laboratories, UK). 2.7. Statistical analysis Data are presented as the mean 7S.E.M. Statistical significance of different groups were performed by one-way analysis of variance followed by Dunnett's test. P o0.05 were considered to indicate statistical significance.
3. Results 3.1. Effects of farrerol on cell viability Cell viability of chondrocytes was assessed by using MTT assay. As shown in Fig. 1, no cytotoxicity of farrerol on chondrocytes was observed at the doses of 0–50 μM. The results showed a reduction
Fig. 2. Farrerol inhibits IL-1β-induced NO and PGE2 production. Chondrocytes were pretreated with farrerol 1 h and then stimulated with IL-1β (10ng/ml) for 24 h. The levels of NO and PGE2 were detected. The data presented are the means 7 S.E.M. of three independent experiments. #P o 0.05 vs. control group; *Po 0.05, **Po 0.01 vs. IL-1β group.
H. Zhang et al. / European Journal of Pharmacology 764 (2015) 443–447
of IL-1β-induced cell viability compared with the control group. Farrerol at doses of 25 and 50 μM reversed the effects of IL-1β on cell viability. Thus, farrerol at the doses of 12.5, 25, 50 μM were used in the subsequent studies. 3.2. Effects of farrerol on IL-1β-induced NO and PGE2 production To investigate the anti-inflammatory effects of farrerol, the effects of farrerol on IL-1β-induced NO and PGE2 production were measured in this study. As shown in Fig. 2, IL-1β significantly upregulated the production of NO and PGE2. However, farrerol dosedependently suppressed IL-1β-induced NO and PGE2 production.
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3.3. Effects of farrerol on IL-1β-induced iNOS and COX-2 expression In this study, the effects of farrerol on IL-1β-induced iNOS and COX-2 expression were detected by western blot analysis. The results showed that compared to the control group, IL-1β remarkably up-regulated the expression of iNOS and COX-2. However, farrerol dose-dependently suppressed IL-1β-induced iNOS and COX-2 expression (Fig. 3). 3.4. Farrerol inhibits IL-1β-induced NF-κB activation It is well known that NF-κB plays an important role in the regulation of inflammatory mediators. To investigate the anti-
Fig. 3. Farrerol inhibits IL-1β-induced iNOS and COX-2 expression. Chondrocytes were pretreated with farrerol 1 h and then stimulated with IL-1β (10 ng/ml) for 24 h. The expression of iNOs and COX-2 were detected by western blot analysis. The data presented are the means 7 S.E.M. of three independent experiments. #P o0.05 vs. control group; *Po 0.05, **P o0.01 vs. IL-1β group.
Fig. 4. Farrerol inhibits IL-1β-induced NF-κB activation and IκBα degradation. Chondrocytes were pretreated with farrerol 1 h and then stimulated with IL-1β (10 ng/ml) for 1 h. NF-κB activation and IκBα degradation were detected by western blot analysis. The values presented are the means 7S.E.M. of three independent experiments. #Po 0.05 vs. control group; *P o0.05, **Po 0.01 vs. IL-1β group.
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inflammatory mechanism of farrerol, the effects of farrerol on IL1β-induced NF-κB activation were detected. The results demonstrated that IL-1β significantly up-regulated NF-κB phosphorylation and IκBα degradation in chondrocytes. And the up-regulation was remarkably inhibited by treatment of farrerol (Fig. 4). 3.5. Farrerol inhibits IL-1β-induced PI3K/Akt phosphorylation To further investigate the anti-inflammatory mechanism of farrerol, the effects of farrerol on IL-1β-induced PI3K/Akt phosphorylation were detected by western blot analysis. As shown in Fig. 5, IL-1β significantly up-regulated the phosphorylation of PI3K and AKT compared to the control group. Treatment of farrerol PI3K/Akt dose-dependently suppressed IL-1β-induced phosphorylation.
4. Discussion Farrerol, a new kind of 2,3-dihydro-flavonoid isolated from rhododendron, has been reported to have therapeutic effects on LPS-induced acute lung injury (Ci et al., 2012). However, the antiinflammatory effects of farrerol on IL-1β-induced inflammation in chondrocytes remain unclear. The present study found that pretreatment with farrerol significantly inhibited IL-1β-induced NO
and PGE2 production, as well as COX-2 and iNOS expression. The anti-inflammatory mechanism of farrerol was mediated via inhibiting IL-1β-induced PI3K and AKT phosphorylation. Osteoarthritis (OA) is a widespread musculoskeletal disease that is characterized by articular cartilage degradation and destruction of cartilage matrix (Loeser, 2009). Pro-inflammatory cytokines such as IL-1β produced by activated chondrocytes are believed to play critical roles in the initiation and development of OA (Moos et al., 2000). Recent studies demonstrated that many natural products had the ability to control or treatment of OA by inhibiting inflammatory mediators production (Ahmed et al., 2005; Shakibaei et al., 2007). In the present study, we found that farrerol inhibited IL-1β-induced NO and PGE2 production in a dose-dependent manner. Next, we investigated whether the inhibitory effect of farrerol on NO and PGE2 production were related to iNOS and COX-2 expression. Our results demonstrated that farrerol inhibited IL-1β-induced NO and PGE2 production by suppressing upstream molecules iNOS and COX-2 expression. In addition, farrerol was found to suppress ConA-induced Th1 and Th2 cytokine production mouse T lymphocytes (Xiong et al., 2013). Farrerol also inhibited LPS-induced TNF-α, IL-6 and IL-8 production (Ci et al., 2012). These studies indicated that farrerol had antiinflammatory effects. Taken together, these results suggested that farrerol had anti-inflammatory effects on IL-1β-induced inflammation in chondrocytes.
Fig. 5. Farrerol inhibits IL-1β-induced PI3K/Akt phosphorylation. Chondrocytes were pretreated with farrerol 1 h and then stimulated with IL-1β (10 ng/ml) for 1 h. PI3K and Akt phosphorylation were detected by western blot analysis. The values presented are the means 7S.E.M. of three independent experiments. #Po 0.05 vs. control group; *Po 0.05, **P o 0.01 vs. IL-1β group.
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NF-κB, an important transcriptional factor, has been reported to play critical roles in inflammatory cytokines production (Blackwell and Christman, 1997; Rai et al., 2014). PI3K and Akt, the upstream molecules of NF-κB, have been demonstrated to play important roles in NF-κB activation (Rahman et al., 2004; Wang et al., 2014). Thus, to investigate the anti-inflammatory mechanisms of farrerol, we detected the effects farrerol on PI3K and Akt phosphorylation and NF-κB activation. The results showed that farrerol inhibited IL-1β-induced inflammation in chondrocytes via inhibition of PI3K/Akt phosphorylation and NF-κB activation. MAPK pathways also play critical roles in the regulation of inflammatory cytokines production (Pawate et al., 2004). Recent studies showed that farrerol inhibited hydrogen peroxide-induced ERK1/2 activation in EA.hy926 cells (Li et al., 2014). Farrerol also inhibited hydrogen peroxide-induced p38 MAPK activation in EA. hy926 cells (Li et al., 2013). It has been reported that there are multiple cross-talk points between PI3K and MAPKs pathways (Aksamitiene et al., 2012). Furthermore, AKT activation has been shown to activate MAPK pathway (Binion et al., 2009). In this study, our results showed that farrerol could inhibit IL-1β-induced PI3K and AKT activation. These results suggested that farrerol might have the ability to inhibit MAPK pathways. Our results were consistent with previous studies. In conclusion, our findings showed that farrerol suppressed IL1β-induced production of NO and PGE2, and expression of iNOS and COX-2 in chondrocytes. Furthermore, our molecular data suggest that farrerol exhibited its anti-inflammatory effects by preventing IL-1β-induced PI3K/Akt phosphorylation.
Conflict of interest statement All authors declare that they have no conflict of interest.
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