Life Sciences 136 (2015) 36–41
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Biochanin A inhibits lipopolysaccharide-induced inflammation in human umbilical vein endothelial cells Xiaodong Ming 1, Mingfeng Ding 1, Bo Zhai, Lei Xiao, Taikui Piao, Ming Liu ⁎ The Fourth Hospital of Harbin Medical University, Harbin, Heilongjiang Province 150001, People's Republic of China
a r t i c l e
i n f o
Article history: Received 20 January 2015 Received in revised form 4 May 2015 Accepted 8 June 2015 Available online 2 July 2015 Keywords: Biochanin A Human umbilical vein endothelial cells Cytokine NF-κB PPAR-γ
a b s t r a c t Aim: Biochanin A, an isoflavone isolated from red clover, cabbage or alfalfa, has been reported to have antiinflammatory activity. However, the effects of biochanin A on vascular inflammation have not been investigated. In this study, we investigate the anti-inflammatory effects of biochanin A on lipopolysaccharide (LPS)-induced inflammatory response in human umbilical vein endothelial cells (HUVEC cells). Main methods: The HUVEC cells were treated with biochanin A for 12 h before exposure to LPS. The expression of ECAMs, including VCAM-1, ICAM-1, E-selectin, NF-κB and PPAR-γ was detected by Western blotting. The expression of cytokines TNF-α and IL-8 was detected by ELISA. Key findings: The results showed that biochanin A inhibited LPS-induced TNF-α and IL-8 production. Meanwhile, biochanin A also suppressed VCAM-1, ICAM-1, and E-selectin expression induced by LPS. We also found that biochanin A inhibited NF-κB activation induced by LPS. Furthermore, biochanin A could activate PPAR-γ and the anti-inflammatory effects of biochanin A can be reversed by GW9662, a specific antagonist for PPAR-γ. Significance: In conclusion, the anti-inflammatory effect of biochanin A is associated with activating PPAR-γ, thereby attenuating NF-κB activation and LPS-induced inflammatory response. These findings suggest that biochanin A may be a therapeutic agent for inflammatory cardiovascular disease. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Vascular inflammation plays an important role in the initiation and progression of atherosclerosis [2,31,35]. LPS, the major constituent of the outer membrane of Gram-negative bacteria, has been reported to be a strong risk factor for vascular inflammation [6,28]. Vascular endothelial cells have been reported to play crucial roles in the development of vascular inflammation [3]. Stimulation of human vascular endothelial cells by LPS could induce NF-κB activation and cytokine TNF-α and IL-8 release and enhance expression of adhesion molecules including ICAM and VCAM [19,26]. Peroxisome proliferator-activated receptor gamma (PPAR-γ), belongs to the nuclear receptor superfamily, that plays important roles in glucose metabolism and energy homeostasis [11]. Recently, it has been reported to play an important role in the regulation of inflammatory responses [16]. Biochanin A, one of the major isoflavonoids in red clover, cabbage or alfalfa, has been reported to have anti-viral, anti-tumor, anti-proliferative and anti-inflammatory activities [21,29]. It has been shown that biochanin A exhibited broad spectrum anti-inflammatory ⁎ Corresponding author. E-mail address: [email protected] (M. Liu). 1 These authors contribute equally to this article.
http://dx.doi.org/10.1016/j.lfs.2015.06.015 0024-3205/© 2015 Elsevier Inc. All rights reserved.
effects [25]. Biochanin A was found to inhibit TNF-α and IL-6 production in LPS-stimulated macrophages [30]. Studies showed that biochanin A protected dopaminergic neurons against LPS-induced damage through inhibition of pro-inflammatory cytokine production [4]. Meanwhile, biochanin A was found to suppress ovalbumin (OVA)-induced airway hyperresponsiveness [20]. However, the anti-inflammatory effect of biochanin A on LPS-stimulated human vascular endothelial cells remains unclear. The objective of this work was to determine the antiinflammatory effects of biochanin A on LPS-stimulated human vascular endothelial cells and to elucidate the potential anti-inflammatory mechanism.
2. Materials and methods 2.1. Chemicals and reagents Biochanin A and LPS (Escherichia coli 055:B5) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies against VCAM-1, ICAM-1, E-selectin, NF-κB, IκBα, and PPAR-γ, and horseradish peroxidase-conjugated (HRP) secondary antibodies were purchased from Santa Cruz Biotechnology (Autogen, Bioclear, UK). Enzymelinked immunosorbent assay (ELISA) kits of TNF-α and IL-8 were
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purchased from Biolegend (CA, USA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
2.2. Cell culture HUVEC cell line was purchased from ATCC (Manassas, VA) and cultured in F12K medium containing 10% fetal bovine serum (FBS), 100 units/ml of penicillin and 100 mg/ml of streptomycin at 37 °C with 5% CO2. In all experiments, cells were incubated in the presence or absence of various concentrations of biochanin A that was always added 12 h prior to LPS (1 μg/ml) treatment.
2.3. Cell viability assay HUVEC cells were plated at a density of 5 × 103 cells/well into 96well plates. The cells were added with 50 μl of biochanin A at different concentrations (0–40 μM) for 12 h, followed by stimulation with 50 μl LPS for 36 h. Then, 20 μl MTT (5 mg/ml) was added to each well for an additional 4 h. The supernatant was removed and the formation of formazan was resolved with 150 μl/well of DMSO. The optical density was measured at 570 nm on a microplate reader (TECAN, Austria).
Fig. 1. Biochanin A inhibits LPS-induced TNF-α and IL-8 production. Cells were pretreated with biochanin A (10, 20, 40 μM) for 12 h and then stimulated with 1 μg/ml LPS for 24 h. Levels of TNF-α and IL-8 in culture supernatants were measured by ELISA. The data presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #p b 0.05 vs. control group; **p b 0.01 vs. LPS group.
2.4. Cytokine assays The levels of TNF-α and IL-8 were measured using sandwich enzyme-linked immunosorbent assay (ELISA) kits (Biolegend, USA) according to the manufacturer's instructions.
Fig. 2. Biochanin A inhibits LPS-induced VCAM-1, ICAM-1, and E-selectin expression in a dose-dependent manner. Cells were pretreated with biochanin A (10, 20, 40 μM) for 12 h and then stimulated with 1 μg/ml LPS for 24 h. The expression of VCAM-1, ICAM-1, and E-selectin was measured by Western blotting. The data presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #p b 0.05 vs. control group; **p b 0.01 vs. LPS group.
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with the ECL Plus Western blotting Detection System (Amersham Life Science, UK). Densitometric analysis was performed with the use of a PDI Imageware System (Bio-Rad, Hercules, CA, USA). 2.6. Cell–cell adhesion assay
Fig. 3. Effects of biochanin A on LPS-induced cell adhesion. Cells were pretreated with biochanin A (10, 20, 40 μM) for 12 h and then stimulated with 1 μg/ml LPS for 24 h. Then the cells were con-incubated with fluorescence-labeled neutrophils. 1 h later, nonadherent cells were removed by washing three times. The fluorescent signals of adherent cells were measured using a fluorescence microplate reader. The data presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #p b 0.05 vs. control group; *p b 0.05, **p b 0.01 vs. LPS group.
2.5. Western blot analysis Total proteins from cells were extracted by M-PER Mammalian Protein Extraction Reagent (Thermo). Protein concentration was detected using BCA method. The proteins were separated on 12% SDSpolyacrylamide gel and transferred onto the PVDF membrane. After blocking the nonspecific site with blocking solution (5% nonfat dry milk), the membrane was incubated with specific primary antibody at 4 °C for 12 h. Subsequently, the membrane was incubated with the secondary antibody at room temperature for 2 h. Blots were then developed
Adherence of neutrophils to endothelial cells was detected by fluorescent labeling of neutrophils as described previously [1]. Briefly, HUVEC cells (1 × 104 cells/well) were seeded in 96-well plates. After growth to confluence, cells were treated with biochanin A and LPS. Neutrophils were labeled with 5 μM Vybrant DiD for 20 min. After washing three times, cells were re-suspended in adhesion medium and added to confluent monolayers of HUVEC cells. 1 h later, nonadherent cells were removed by washing three times. The fluorescent signals of adherent cells were measured using a fluorescence microplate reader (Tecan Austria GmbH). The percentage of adherent neutrophils was calculated by adherent signal / total signal × 100%. 2.7. Statistical analysis Data are presented as mean ± standard error of measurement (SEM). Comparison between groups was made with ANOVA followed by Dunnett's test. p-Values of 0.05 or less were considered statistically significant. 3. Results 3.1. Biochanin A inhibits TNF-α and IL-8 production in LPS-stimulated HUVEC cells Inflammatory cytokines and chemokines, such as TNF-α and IL-8, have been reported to play critical roles in the development of
Fig. 4. Biochanin A inhibits LPS-induced NF-κB activation and IκBα degradation. β-Actin was used as a control. Cells were pretreated with biochanin A (10, 20, 40 μM) for 12 h and then stimulated with 1 μg/ml LPS for 1 h. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. # p b 0.05 vs. control group; **p b 0.01 vs. LPS group.
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Fig. 3, biochanin A inhibited LPS-induced adhesion of neutrophils to LPS-stimulated endothelial cells. 3.4. Biochanin A suppresses LPS-induced NF-κB activation NF-κB has been reported to play an important role in the regulation of inflammatory mediator production. In this study, we determined the effects of biochanin A on LPS-induced NF-κB activation. As shown in Fig. 4, LPS could induce NF-κB activation and IκBα degradation. However, biochanin A significantly inhibited the activation of NF-κB and the degradation of IκBα induced by LPS. 3.5. Effects of biochanin A on cell viability The potential cytotoxicity of biochanin A was evaluated by the MTT assay. As shown in Fig. 5, cell viabilities were not affected by biochanin A at concentrations used (10, 20, 40 μM) (Fig. 5A). Biochanin A showed no toxicity to HUVEC cells at these concentrations. 3.6. Effects of biochanin A on PPAR-γ activation PPAR-γ has been reported to have anti-inflammatory effects. In this study, we investigated the effects of biochanin A on PPAR-γ activation. As shown in Fig. 5, our results showed that biochanin A up-regulated PPAR-γ expression in a dose dependent manner (Fig. 5B). 3.7. Effects of biochanin A on LPS-induced inflammatory response in HUVEC cells are PPAR-γ dependent
Fig. 5. (A) Effects of biochanin A on the cell viability of HUVEC cells. Cells were cultured with different concentrations of biochanin A (0–40 μM) in the absence or presence of LPS for 48 h. The cell viability was determined by MTT assay. (B) Effects of biochanin A on PPAR-γ expression. Cells were treated with biochanin A (10, 20, 40 μM) for 12 h. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. *p b 0.05, **p b 0.01 vs. control group.
inflammation. Therefore, to analyze the potential anti-inflammatory effect of biochanin A in LPS-stimulated HUVEC cells, TNF-α and IL-8 production was determined. As shown in Fig. 1, the production of TNF-α and IL-8 increased significantly when HUVEC cells were stimulated by LPS. Biochanin A was found to inhibit TNF-α and IL-8 production in LPS-stimulated HUVEC cells in a dose-dependent manner (Fig. 1). 3.2. Biochanin A inhibits VCAM-1, ICAM-1, and E-selectin expression in LPSstimulated HUVEC cells Adhesion molecules such as VCAM-1, ICAM-1, and E-selectin, play a major role in the formation of firm adhesion and transendothelial migration of leucocytes into inflamed vessels. In this study, the effects of biochanin A on LPS-induced VCAM-1, ICAM-1, and E-selectin expression were determined by Western blotting. As shown in Fig. 2, LPS significantly increased VCAM-1, ICAM-1, and E-selectin expression in HUVEC cells. However, the levels of VCAM-1, ICAM-1, and E-selectin induced by LPS were inhibited by biochanin A in a dose dependent manner. 3.3. Biochanin A inhibits adhesion of neutrophils to LPS-stimulated endothelial cells The adhesion of neutrophils to the endothelium is a crucial step in the inflammatory response which precedes the development of atherosclerosis. In this study, we determined the effects of biochanin A on the adhesion of neutrophils to LPS-stimulated endothelial cells. As shown in
To detect the anti-inflammatory mechanism of biochanin A, GW9662, the PPAR-γ specific antagonist was used in this study. As shown in Fig. 6, biochanin A significantly inhibited TNF-α and IL-8 production, as well as NF-κB activation. However, once PPAR-γ was inhibited by GW9662, the anti-inflammatory effects of biochanin A were abolished. 4. Discussion Vascular inflammatory responses play important roles in the pathogenesis of atherosclerosis [5]. Endothelial cells play an important role in vascular inflammatory process [27]. Biochanin A, one of the major isoflavonoids in red clover, cabbage or alfalfa, has been reported to have anti-inflammatory effects. In this study, the anti-inflammatory effect of biochanin A on LPS-stimulated HUVEC cells was assayed. The results showed that the anti-inflammatory effect of biochanin A was PPAR-γ dependent. Inflammation plays an important role in the development of atherosclerosis [15]. LPS, the major constituent of the outer membrane of Gram-negative bacteria, is one of the strongest stimulators that targets the endothelium [17]. LPS could induce the expression of cytokines TNF-α and IL-8 [10,38]. IL-8 is a monocyte chemoattractant cytokine which could recruit monocytes into the tunica intima. TNF-α could induce the production of other inflammatory cytokines which may irreversibly damage vascular integrity [18,22]. Therefore, treatments aimed at suppressing pro-inflammatory mediators have potential therapeutic strategy for atherosclerosis. The results demonstrated that biochanin A inhibited the expression of TNF-α and IL-8 in LPS-stimulated human vascular endothelial cells in a dose-dependent manner. The adhesion of neutrophils to the endothelium is a crucial step in the inflammatory response which precedes the development of atherosclerosis [12]. VCAM-1, ICAM-1 and E-selectin recruit leukocytes to the sites of inflammation [36]. They also play an important role in the inflammatory response. In this study, we found that biochanin A inhibited LPS-induced adhesion of neutrophils to LPS-stimulated endothelial cells. Meanwhile, biochanin A suppressed the up-regulation of VCAM-1, ICAM-1 and E-selectin induced by LPS.
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Fig. 6. Anti-inflammatory effects of biochanin A on LPS-induce inflammatory response are PPAR-γ dependent. Cells were treated with 40 μM biochanin A for 12 h, or 10 μM GW9662 for 30 min before biochanin A incubation, and stimulated with LPS for 24 h. Levels of TNF-α and IL-8 were detected by ELISA. The values presented are the means ± SEM of three independent experiments. #p b 0.05 vs. control group; *p b 0.05, **p b 0.01 vs. LPS group.
NF-κB is the primary transcription factor which is involved in the inflammatory responses [9,23]. It is well known that NF-κB is involved in the regulation of VCAM-1, ICAM-1 and E-selectin expression and cytokine production [33,34]. Once stimulated with LPS, NF-κB is activated by phosphorylation and regulates the expression of a network of inflammatory mediators [7,13,37]. Thus, we detected the effects of biochanin A on LPS-induced NF-κB activation. Our results demonstrated that biochanin A inhibited LPS-induced pro-inflammatory mediator production by suppressing NF-κB activation. PPAR-γ, a member of the nuclear hormone receptor superfamily, has been reported to play a critical role in adipocyte differentiation and glucose homeostasis [8]. Recently, it has been shown to have an anti-inflammatory effect. Activation of PPAR-γ has been reported to inhibit NF-κB activation and inflammatory mediator production [14]. Some reports have demonstrated that many herbal products exerted anti-inflammatory effects by activating PPAR-γ [24,32]. In this study, our results showed that biochanin A up-regulated PPAR-γ expression in a dose-dependent manner. Inhibiting PPAR-γ by GW9662, the anti-inflammatory effects of biochanin A were abolished.
5. Conclusion Our results demonstrate that biochanin A inhibits inflammatory mediator expression in LPS-stimulated human vascular endothelial cells. The promising anti-inflammatory effect of biochanin A is associated with up-regulation of PPAR-γ, thereby attenuating NF-κB activation and LPS-induced inflammatory response.
Author contributions X.D.M., M.F.D., and M.L. contributed to the conception and design and wrote the article. B.Z., L.X. and T.K.P. did the analysis and interpretation.
Conflict of interest The authors have no conflict of interest to declare.
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Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
Biochanin A inhibits lipopolysaccharide-induced inflammation in human umbilical vein endothelial cells Xiaodong Ming 1, Mingfeng Ding 1, Bo Zhai, Lei Xiao, Taikui Piao, Ming Liu ⁎ The Fourth Hospital of Harbin Medical University, Harbin, Heilongjiang Province 150001, People's Republic of China
a r t i c l e
i n f o
Article history: Received 20 January 2015 Received in revised form 4 May 2015 Accepted 8 June 2015 Available online 2 July 2015 Keywords: Biochanin A Human umbilical vein endothelial cells Cytokine NF-κB PPAR-γ
a b s t r a c t Aim: Biochanin A, an isoflavone isolated from red clover, cabbage or alfalfa, has been reported to have antiinflammatory activity. However, the effects of biochanin A on vascular inflammation have not been investigated. In this study, we investigate the anti-inflammatory effects of biochanin A on lipopolysaccharide (LPS)-induced inflammatory response in human umbilical vein endothelial cells (HUVEC cells). Main methods: The HUVEC cells were treated with biochanin A for 12 h before exposure to LPS. The expression of ECAMs, including VCAM-1, ICAM-1, E-selectin, NF-κB and PPAR-γ was detected by Western blotting. The expression of cytokines TNF-α and IL-8 was detected by ELISA. Key findings: The results showed that biochanin A inhibited LPS-induced TNF-α and IL-8 production. Meanwhile, biochanin A also suppressed VCAM-1, ICAM-1, and E-selectin expression induced by LPS. We also found that biochanin A inhibited NF-κB activation induced by LPS. Furthermore, biochanin A could activate PPAR-γ and the anti-inflammatory effects of biochanin A can be reversed by GW9662, a specific antagonist for PPAR-γ. Significance: In conclusion, the anti-inflammatory effect of biochanin A is associated with activating PPAR-γ, thereby attenuating NF-κB activation and LPS-induced inflammatory response. These findings suggest that biochanin A may be a therapeutic agent for inflammatory cardiovascular disease. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Vascular inflammation plays an important role in the initiation and progression of atherosclerosis [2,31,35]. LPS, the major constituent of the outer membrane of Gram-negative bacteria, has been reported to be a strong risk factor for vascular inflammation [6,28]. Vascular endothelial cells have been reported to play crucial roles in the development of vascular inflammation [3]. Stimulation of human vascular endothelial cells by LPS could induce NF-κB activation and cytokine TNF-α and IL-8 release and enhance expression of adhesion molecules including ICAM and VCAM [19,26]. Peroxisome proliferator-activated receptor gamma (PPAR-γ), belongs to the nuclear receptor superfamily, that plays important roles in glucose metabolism and energy homeostasis [11]. Recently, it has been reported to play an important role in the regulation of inflammatory responses [16]. Biochanin A, one of the major isoflavonoids in red clover, cabbage or alfalfa, has been reported to have anti-viral, anti-tumor, anti-proliferative and anti-inflammatory activities [21,29]. It has been shown that biochanin A exhibited broad spectrum anti-inflammatory ⁎ Corresponding author. E-mail address: [email protected] (M. Liu). 1 These authors contribute equally to this article.
http://dx.doi.org/10.1016/j.lfs.2015.06.015 0024-3205/© 2015 Elsevier Inc. All rights reserved.
effects [25]. Biochanin A was found to inhibit TNF-α and IL-6 production in LPS-stimulated macrophages [30]. Studies showed that biochanin A protected dopaminergic neurons against LPS-induced damage through inhibition of pro-inflammatory cytokine production [4]. Meanwhile, biochanin A was found to suppress ovalbumin (OVA)-induced airway hyperresponsiveness [20]. However, the anti-inflammatory effect of biochanin A on LPS-stimulated human vascular endothelial cells remains unclear. The objective of this work was to determine the antiinflammatory effects of biochanin A on LPS-stimulated human vascular endothelial cells and to elucidate the potential anti-inflammatory mechanism.
2. Materials and methods 2.1. Chemicals and reagents Biochanin A and LPS (Escherichia coli 055:B5) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies against VCAM-1, ICAM-1, E-selectin, NF-κB, IκBα, and PPAR-γ, and horseradish peroxidase-conjugated (HRP) secondary antibodies were purchased from Santa Cruz Biotechnology (Autogen, Bioclear, UK). Enzymelinked immunosorbent assay (ELISA) kits of TNF-α and IL-8 were
X. Ming et al. / Life Sciences 136 (2015) 36–41
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purchased from Biolegend (CA, USA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
2.2. Cell culture HUVEC cell line was purchased from ATCC (Manassas, VA) and cultured in F12K medium containing 10% fetal bovine serum (FBS), 100 units/ml of penicillin and 100 mg/ml of streptomycin at 37 °C with 5% CO2. In all experiments, cells were incubated in the presence or absence of various concentrations of biochanin A that was always added 12 h prior to LPS (1 μg/ml) treatment.
2.3. Cell viability assay HUVEC cells were plated at a density of 5 × 103 cells/well into 96well plates. The cells were added with 50 μl of biochanin A at different concentrations (0–40 μM) for 12 h, followed by stimulation with 50 μl LPS for 36 h. Then, 20 μl MTT (5 mg/ml) was added to each well for an additional 4 h. The supernatant was removed and the formation of formazan was resolved with 150 μl/well of DMSO. The optical density was measured at 570 nm on a microplate reader (TECAN, Austria).
Fig. 1. Biochanin A inhibits LPS-induced TNF-α and IL-8 production. Cells were pretreated with biochanin A (10, 20, 40 μM) for 12 h and then stimulated with 1 μg/ml LPS for 24 h. Levels of TNF-α and IL-8 in culture supernatants were measured by ELISA. The data presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #p b 0.05 vs. control group; **p b 0.01 vs. LPS group.
2.4. Cytokine assays The levels of TNF-α and IL-8 were measured using sandwich enzyme-linked immunosorbent assay (ELISA) kits (Biolegend, USA) according to the manufacturer's instructions.
Fig. 2. Biochanin A inhibits LPS-induced VCAM-1, ICAM-1, and E-selectin expression in a dose-dependent manner. Cells were pretreated with biochanin A (10, 20, 40 μM) for 12 h and then stimulated with 1 μg/ml LPS for 24 h. The expression of VCAM-1, ICAM-1, and E-selectin was measured by Western blotting. The data presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #p b 0.05 vs. control group; **p b 0.01 vs. LPS group.
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with the ECL Plus Western blotting Detection System (Amersham Life Science, UK). Densitometric analysis was performed with the use of a PDI Imageware System (Bio-Rad, Hercules, CA, USA). 2.6. Cell–cell adhesion assay
Fig. 3. Effects of biochanin A on LPS-induced cell adhesion. Cells were pretreated with biochanin A (10, 20, 40 μM) for 12 h and then stimulated with 1 μg/ml LPS for 24 h. Then the cells were con-incubated with fluorescence-labeled neutrophils. 1 h later, nonadherent cells were removed by washing three times. The fluorescent signals of adherent cells were measured using a fluorescence microplate reader. The data presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #p b 0.05 vs. control group; *p b 0.05, **p b 0.01 vs. LPS group.
2.5. Western blot analysis Total proteins from cells were extracted by M-PER Mammalian Protein Extraction Reagent (Thermo). Protein concentration was detected using BCA method. The proteins were separated on 12% SDSpolyacrylamide gel and transferred onto the PVDF membrane. After blocking the nonspecific site with blocking solution (5% nonfat dry milk), the membrane was incubated with specific primary antibody at 4 °C for 12 h. Subsequently, the membrane was incubated with the secondary antibody at room temperature for 2 h. Blots were then developed
Adherence of neutrophils to endothelial cells was detected by fluorescent labeling of neutrophils as described previously [1]. Briefly, HUVEC cells (1 × 104 cells/well) were seeded in 96-well plates. After growth to confluence, cells were treated with biochanin A and LPS. Neutrophils were labeled with 5 μM Vybrant DiD for 20 min. After washing three times, cells were re-suspended in adhesion medium and added to confluent monolayers of HUVEC cells. 1 h later, nonadherent cells were removed by washing three times. The fluorescent signals of adherent cells were measured using a fluorescence microplate reader (Tecan Austria GmbH). The percentage of adherent neutrophils was calculated by adherent signal / total signal × 100%. 2.7. Statistical analysis Data are presented as mean ± standard error of measurement (SEM). Comparison between groups was made with ANOVA followed by Dunnett's test. p-Values of 0.05 or less were considered statistically significant. 3. Results 3.1. Biochanin A inhibits TNF-α and IL-8 production in LPS-stimulated HUVEC cells Inflammatory cytokines and chemokines, such as TNF-α and IL-8, have been reported to play critical roles in the development of
Fig. 4. Biochanin A inhibits LPS-induced NF-κB activation and IκBα degradation. β-Actin was used as a control. Cells were pretreated with biochanin A (10, 20, 40 μM) for 12 h and then stimulated with 1 μg/ml LPS for 1 h. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. # p b 0.05 vs. control group; **p b 0.01 vs. LPS group.
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Fig. 3, biochanin A inhibited LPS-induced adhesion of neutrophils to LPS-stimulated endothelial cells. 3.4. Biochanin A suppresses LPS-induced NF-κB activation NF-κB has been reported to play an important role in the regulation of inflammatory mediator production. In this study, we determined the effects of biochanin A on LPS-induced NF-κB activation. As shown in Fig. 4, LPS could induce NF-κB activation and IκBα degradation. However, biochanin A significantly inhibited the activation of NF-κB and the degradation of IκBα induced by LPS. 3.5. Effects of biochanin A on cell viability The potential cytotoxicity of biochanin A was evaluated by the MTT assay. As shown in Fig. 5, cell viabilities were not affected by biochanin A at concentrations used (10, 20, 40 μM) (Fig. 5A). Biochanin A showed no toxicity to HUVEC cells at these concentrations. 3.6. Effects of biochanin A on PPAR-γ activation PPAR-γ has been reported to have anti-inflammatory effects. In this study, we investigated the effects of biochanin A on PPAR-γ activation. As shown in Fig. 5, our results showed that biochanin A up-regulated PPAR-γ expression in a dose dependent manner (Fig. 5B). 3.7. Effects of biochanin A on LPS-induced inflammatory response in HUVEC cells are PPAR-γ dependent
Fig. 5. (A) Effects of biochanin A on the cell viability of HUVEC cells. Cells were cultured with different concentrations of biochanin A (0–40 μM) in the absence or presence of LPS for 48 h. The cell viability was determined by MTT assay. (B) Effects of biochanin A on PPAR-γ expression. Cells were treated with biochanin A (10, 20, 40 μM) for 12 h. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. *p b 0.05, **p b 0.01 vs. control group.
inflammation. Therefore, to analyze the potential anti-inflammatory effect of biochanin A in LPS-stimulated HUVEC cells, TNF-α and IL-8 production was determined. As shown in Fig. 1, the production of TNF-α and IL-8 increased significantly when HUVEC cells were stimulated by LPS. Biochanin A was found to inhibit TNF-α and IL-8 production in LPS-stimulated HUVEC cells in a dose-dependent manner (Fig. 1). 3.2. Biochanin A inhibits VCAM-1, ICAM-1, and E-selectin expression in LPSstimulated HUVEC cells Adhesion molecules such as VCAM-1, ICAM-1, and E-selectin, play a major role in the formation of firm adhesion and transendothelial migration of leucocytes into inflamed vessels. In this study, the effects of biochanin A on LPS-induced VCAM-1, ICAM-1, and E-selectin expression were determined by Western blotting. As shown in Fig. 2, LPS significantly increased VCAM-1, ICAM-1, and E-selectin expression in HUVEC cells. However, the levels of VCAM-1, ICAM-1, and E-selectin induced by LPS were inhibited by biochanin A in a dose dependent manner. 3.3. Biochanin A inhibits adhesion of neutrophils to LPS-stimulated endothelial cells The adhesion of neutrophils to the endothelium is a crucial step in the inflammatory response which precedes the development of atherosclerosis. In this study, we determined the effects of biochanin A on the adhesion of neutrophils to LPS-stimulated endothelial cells. As shown in
To detect the anti-inflammatory mechanism of biochanin A, GW9662, the PPAR-γ specific antagonist was used in this study. As shown in Fig. 6, biochanin A significantly inhibited TNF-α and IL-8 production, as well as NF-κB activation. However, once PPAR-γ was inhibited by GW9662, the anti-inflammatory effects of biochanin A were abolished. 4. Discussion Vascular inflammatory responses play important roles in the pathogenesis of atherosclerosis [5]. Endothelial cells play an important role in vascular inflammatory process [27]. Biochanin A, one of the major isoflavonoids in red clover, cabbage or alfalfa, has been reported to have anti-inflammatory effects. In this study, the anti-inflammatory effect of biochanin A on LPS-stimulated HUVEC cells was assayed. The results showed that the anti-inflammatory effect of biochanin A was PPAR-γ dependent. Inflammation plays an important role in the development of atherosclerosis [15]. LPS, the major constituent of the outer membrane of Gram-negative bacteria, is one of the strongest stimulators that targets the endothelium [17]. LPS could induce the expression of cytokines TNF-α and IL-8 [10,38]. IL-8 is a monocyte chemoattractant cytokine which could recruit monocytes into the tunica intima. TNF-α could induce the production of other inflammatory cytokines which may irreversibly damage vascular integrity [18,22]. Therefore, treatments aimed at suppressing pro-inflammatory mediators have potential therapeutic strategy for atherosclerosis. The results demonstrated that biochanin A inhibited the expression of TNF-α and IL-8 in LPS-stimulated human vascular endothelial cells in a dose-dependent manner. The adhesion of neutrophils to the endothelium is a crucial step in the inflammatory response which precedes the development of atherosclerosis [12]. VCAM-1, ICAM-1 and E-selectin recruit leukocytes to the sites of inflammation [36]. They also play an important role in the inflammatory response. In this study, we found that biochanin A inhibited LPS-induced adhesion of neutrophils to LPS-stimulated endothelial cells. Meanwhile, biochanin A suppressed the up-regulation of VCAM-1, ICAM-1 and E-selectin induced by LPS.
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Fig. 6. Anti-inflammatory effects of biochanin A on LPS-induce inflammatory response are PPAR-γ dependent. Cells were treated with 40 μM biochanin A for 12 h, or 10 μM GW9662 for 30 min before biochanin A incubation, and stimulated with LPS for 24 h. Levels of TNF-α and IL-8 were detected by ELISA. The values presented are the means ± SEM of three independent experiments. #p b 0.05 vs. control group; *p b 0.05, **p b 0.01 vs. LPS group.
NF-κB is the primary transcription factor which is involved in the inflammatory responses [9,23]. It is well known that NF-κB is involved in the regulation of VCAM-1, ICAM-1 and E-selectin expression and cytokine production [33,34]. Once stimulated with LPS, NF-κB is activated by phosphorylation and regulates the expression of a network of inflammatory mediators [7,13,37]. Thus, we detected the effects of biochanin A on LPS-induced NF-κB activation. Our results demonstrated that biochanin A inhibited LPS-induced pro-inflammatory mediator production by suppressing NF-κB activation. PPAR-γ, a member of the nuclear hormone receptor superfamily, has been reported to play a critical role in adipocyte differentiation and glucose homeostasis [8]. Recently, it has been shown to have an anti-inflammatory effect. Activation of PPAR-γ has been reported to inhibit NF-κB activation and inflammatory mediator production [14]. Some reports have demonstrated that many herbal products exerted anti-inflammatory effects by activating PPAR-γ [24,32]. In this study, our results showed that biochanin A up-regulated PPAR-γ expression in a dose-dependent manner. Inhibiting PPAR-γ by GW9662, the anti-inflammatory effects of biochanin A were abolished.
5. Conclusion Our results demonstrate that biochanin A inhibits inflammatory mediator expression in LPS-stimulated human vascular endothelial cells. The promising anti-inflammatory effect of biochanin A is associated with up-regulation of PPAR-γ, thereby attenuating NF-κB activation and LPS-induced inflammatory response.
Author contributions X.D.M., M.F.D., and M.L. contributed to the conception and design and wrote the article. B.Z., L.X. and T.K.P. did the analysis and interpretation.
Conflict of interest The authors have no conflict of interest to declare.
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